Establishing an Adult Mouse Brain Hippocampal Organotypic Slice Culture System that Allows for Tracing and Pharmacological Manipulation of ex vivo Neurogenesis
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Author:
Date:
2021-01-05
[Abstract] The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the ...
[摘要] [摘要]海马的功能取决于成年海马神经发生的过程,该过程是这种结构异常的神经发育的基础,并且在中枢神经系统病理中也经常受到影响。因此,对该过程的操纵代表了重要的治疗目标。为了确定潜在的策略,器官型成人大脑切片正在成为一种有价值的工具。近年来,此方法已得到完善,在此我们提出一种组合协议, 汇集了这些改进,以实现成人海马切片的长期培养。我们采用了一种切片技术,可将必要的传入输入保留在海马上以及无血清培养条件下,因此可以延长培养时间。为了维持切片中的神经源性潜力,我们利用神经胶质生成抑制剂吲哚美辛。使用EdU保留分析使我们能够评估药理干预对神经发生的影响。通过这些改进,我们建立了一种简单可靠的方法来研究小分子/药物对离体增殖和神经元形成的影响,这将有助于未来发现驱动的药物筛选。
[背景技术]海马是具有高度的可塑性作为整个生命齿状回中正在进行的神经发生的结果,脑的独特区域。成年海马神经发生的这一过程始于在亚颗粒区(SGZ)中神经干细胞(NSC)的不对称分裂,该过程保留了干细胞池并生成了准备用于神经元分化的祖细胞(Kempermann等人,2004;Anacker和Hen ,2017; ...
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Brain Tissue Culture of Per2::Luciferase Transgenic Mice for ex vivo Bioluminescence
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Author:
Date:
2018-07-05
[Abstract] In circadian research, it is essential to be able to track a biological rhythm for several days with the minimum perturbation for the organisms or tissues. The use of transgenic mice lines, in which the luciferase reporter is coupled to a molecular clock protein (here PERIOD2), gives us the opportunity to follow the circadian activity in different tissues or even single clock cells for days without manipulation. This method creates sections using a mouse brain matrix, which allows us to obtain several brain samples quickly at a single time point.
[摘要] 在昼夜节律研究中,能够以最小的生物或组织扰动跟踪生物节律数天是至关重要的。 使用转基因小鼠系,其中荧光素酶报告基因与分子钟蛋白(此处为PERIOD2)偶联,使我们有机会在不经操作的情况下跟踪不同组织或甚至单个时钟细胞中的昼夜节律活动数天。 该方法使用鼠标脑矩阵创建切片,这允许我们在单个时间点快速获得几个脑样本。
【背景】昼夜节律是大约24小时循环的行为或分子变化,并且在没有任何外部线索的情况下持续。在哺乳动物中,运动活动,体温和激素释放是昼夜节律的实例,其在位于下丘脑的视交叉上核(SCN)时钟的控制下。 SCN细胞保持内源性节律的能力是由于时钟基因表达的正负循环组成的分子机制:首先,CLOCK和BMAL1蛋白异二聚化通过E-box位点激活不同基因的转录关于基因如 period ( Per1-3 )和隐花色素( Cry1-2 ; Takahashi)的启动子 et al。,2008)。然后,PERIOD和CRYPTOCHROME的蛋白质异二聚化并返回到细胞核以防止BMAL1与E-Box结合。因此,PERIOD和CRYPTOCHROME抑制其自身的转录(Takahashi et al。,2008)。第二个环由类视黄醇相关的孤儿受体(ROR)和Rev-Erb组成:ROR蛋白激活 Bmal1 基因,而REV-ERB蛋白抑制它通过 ROR反应 Bmal1 ...
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