| Imaging Microtubules in vitro at High Resolution while Preserving their Structure
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Author:
Date:
2021-04-05
[Abstract] Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can ...
[摘要] [摘要]微管（MT）是细胞骨架中最刚性的组成部分。然而，它们在细胞环境中经常显得高度弯曲，并且控制它们整体形状的机理了解甚少。当前，体外微管分析主要依靠电子显微镜进行高分辨率分析，而全内反射荧光（TIRF ）显微镜则可以对活的荧光标记的微管和相关蛋白进行成像。为了对具有微米曲率的微管进行三维分析，我们开发了一种在体外聚合MT的检测方法 用类似于常规TIRF显微镜操作规程的方式将MT种子的MT粘附到载玻片上。除去游离的荧光分子，并通过灌注固定MTs。然后可以使用带有Airyscan模块的共聚焦显微镜观察MT，以获得更高的分辨率。该协议允许对保留其原始三维形状并与高分辨率免疫荧光检测兼容的微管进行成像。
[背景]微管（MT）是通过异源二聚体的组合制成的聚合物α和β微管蛋白，并且是细胞骨架的主要成分。他们参与了细胞功能的基本机制，如有丝分裂，细胞内转运，胞质分裂和细胞形态的维持（Akhmanova和Steinmetz，2015）。尽管MT本身具有很高的刚性，但它们通常会在细胞中弯曲并产生一些蛋白，从而弯曲微管（Brangwynne等人，2006; Bechstedt等人，2014; Leung等人，2020; Cuveillier等人，2020 ...
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| Characterization of Hippocampal Adult-borne Granule Cells in a Transient Cerebral Ischemia Model
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Author:
Date:
2021-01-20
[Abstract] Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne ...
[摘要] [摘要]中风的长期后果严重损害了越来越多的中风幸存者的生活质量。假定海马成年神经发生在中风的认知和神经精神病学长期后遗症的病理生理学中起作用。可靠的中风动物模型对于理解其生物机制和推进治疗策略至关重要。我们提出 暂时性脑缺血模型的详细协议，该模型不会在海马体中引起直接缺血损伤，从而可以研究中风的长期神经认知缺陷的病理生理学。此外，我们描述了一种用于获取成人海马颗粒细胞的电生理学和形态学特征的急性海马切片的协议。还讨论了与从小细胞（例如未成熟的成年颗粒细胞）进行电生理记录有关的特殊性。本协议可以通过多模式研究（行为，形态结构，生化）加以补充，以期有希望促进研究和中风的长期后遗症的发展以及发现新的治疗机会。
[背景]中风是在发达国家发病率和死亡率的重要原因引起急性和延迟赤字。虽然针对缺血性中风恢复血流的干预策略在降低急性发病率和死亡率方面已经变得更加有效，但中风的长期后果（如中风后抑郁和中风后认知功能障碍和痴呆症）目前正在逃避临床治疗（Wang等。，2010; Loubinoux等人，2012; Mijajlovic ...
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| Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain
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Author:
Date:
2020-09-05
[Abstract] Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish ...
[摘要] [摘要] 人类神经元移植为建模人类神经系统疾病和潜在的替代疗法提供了新的机会。然而，人脑皮层的复杂结构分为六层，具有紧密互连的兴奋性和抑制性神经元网络，这对体内移植技术获得平衡，功能稳定和稳态稳定的神经元网络提出了重大挑战。在这里，我们提出了一项协议，将人类诱导的多能干细胞（hiPSC ）衍生的神经祖细胞引入大鼠脑。使用这种方法，hiPSC 衍生的神经元在结构上整合到大鼠前脑中，表现出电生理特性，包括放电，兴奋性和抑制性突触活性，并与宿主电路建立神经元连通性。
[背景] 人类大脑皮层是一个复杂的细胞镶嵌体，在不同的皮质层（I-VI）中包含多样化的神经元亚型，可建立轴突输出和树突状输入的特定模式，提供了皮质电路的基本底物（Rakic，2009; Lodato 等等人，2011； Lui 等人，2011）。特别地，兴奋性和抑制性神经传递的平衡对于适当的脑功能是必需的（Turrigiano和Nelson，2004）。人类诱导的多能干细胞（hiPSC ）可以在人类遗传背景下对人类神经系统疾病进行建模（Dolmetsch和Geschwind，2011； Brennand 等，2015； Vera和Studer，2015）。建立体外系统以将hiPSCs ...
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