| Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
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Author:
Date:
2021-04-20
[Abstract] Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a ...
[摘要] [摘要]参与神经变性蛋白质可具有耦合光遗传学试剂来创建快速且灵敏的记者到provid Ë洞察介导的神经变性疾病，包括进展的生化过程阿尔茨海默氏病（AD）。我们最近开发了一种新型光学-响应工具（“辅”系统）夫妇COF伊林和行动中使用（与神经退行性疾病相关的早期阶段，细胞骨架异常关键球员）光门控光遗传学 蛋白质提供时空分辨率的氧化和高能应激依赖的生化事件。与目前可用的基于小分子的生物传感器来监测细胞氧化还原环境的变化相比，CofActor是一种光激活的，遗传编码的氧化还原传感器，可以通过精确的空间和时间控制来激活。在这里，我们描述了从新生小鼠制备的解离海马神经元培养物中CofActor系统的表达和激活的协议。将培养物转染用大号ipofectamine上的第五天体外（DIV5），然后暴露于细胞应激诱导刺激，导致的肌动蛋白的形成丝切蛋白可使用活细胞成像技术可以观察到杆。本文所述的方案可用于研究暴露于神经退行性刺激（例如毒性Aβ42低聚物）的活神经元中与压力相关的细胞骨架失调。此外，从AD的转基因小鼠模型和/或与KO相关的小鼠KO小鼠分离的神经元中传感器的表达可以促进我们对与神经变性相关的早期细胞骨架功能障碍的分子基础的理解。
[背景]神经变性疾病的生化标志（神经原纤维，团块和缠结，提高活性氧物质（ROS） ...
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| Craniotomy for Cortical Voltage-sensitive Dye Imaging in Mice
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Author:
Date:
2016-02-05
[Abstract] Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image sensor with a fast sampling rate (500 Hz) detects fluorescent changes in corrective activity. The procedure includes fixing a mouse brain to a stereotaxic apparatus, craniotomy on a large cortical area, VSD staining, and wide-field imaging of cortical activity. The entire procedure can be completed in 5 h (from the administration of anesthesia to the start of cortical VSD imaging).
[摘要] 皮层皮层相互作用在各种大脑功能中起关键作用。 在这里，我们提供皮质电压敏感染料（VSD）成像允许监测活体小鼠皮质活动的时空动力学的详细的外科手术。 上层（层1-3）中的皮层神经元用VSD染色，并且具有快速采样率（500Hz）的图像传感器检测校正活性中的荧光变化。 该程序包括将小鼠脑固定到立体定位装置，在大皮质区域上的颅骨切开术，VSD染色和皮层活动的宽视野成像。 整个过程可以在5小时内完成（从麻醉的施用到皮质VSD成像的开始）。
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| Retinal Explant Culture
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Author:
Date:
2014-01-20
[Abstract] A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from ...
[摘要] 视网膜的特别强大的培养方法是外植体测定，其包括在器官型过滤器上培养小片视网膜。视网膜外植体可以在胚胎第13天（E13）和出生后第4天（P4）之间的任何时间进行。虽然视网膜神经节细胞在外植体中产生后不久就会退化，并且感光细胞不会扩展外延伸的外切片，外植体将在体内与视网膜非常相似，并产生将迁移到适当的所有不同的视网膜细胞类型层。视网膜外植体培养测定在小鼠突变体是胚胎致死性并且其视网膜发育不能在体内研究的情况下特别有用。因为视网膜外植体可以从胚胎动物制备并用病毒载体电穿孔或感染，因此也是胚胎期基因功能研究的有用途径。在这里，我们提出了我们在各种出版物中广泛使用的视网膜外植体培养方法（Kechad等，2012; Cayouette等，2003; Cayouette和Raff，2003; Elliott等，2008）。
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