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Potassium chloride

氯化钾

Company: Sigma-Aldrich
Catalog#: P3911
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Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia
Author:
Date:
2018-03-05
[Abstract]  In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems. [摘要]  在这个协议中,我们介绍了一种有效的方法,用于Tomina和Wagenaar(2017)中使用的电压敏感染料(VSD)加载和水蛭神经节成像。 解剖和染料加载程序是成功完成全神经节VSD成像的关键步骤。 前者需要去除覆盖水蛭节段神经节神经元的鞘,这是成功染料加载所需的。 后者需要使用一对蠕动泵同时轻柔地将新一代VSD VF2.1(OMe).H流入神经节的两侧。 我们期望所描述的技术广泛地转化为其他薄且相对透明的神经系统中的宽视场VSD成像。

【背景】双面显微镜是一种宽视野荧光成像系统,由一对精确对准的显微镜组成,用于观察来自对面的神经元制剂并且一次显示不同的焦平面(Tomina and Wagenaar,2017)。通过将该光学系统与新一代电压敏感染料(VSD),VoltageFluor(Miller等人,2012; Woodford等人,2015),荧光可以同时从不同深度的神经元捕获编码具有高保真度膜电压的信号。我们将这种泛神经元记录系统应用于药用水蛭的神经系统,我们利用电生理学方法诱发虚构行为并定量控制可识别神经元的膜电位(Tomina and ...

High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
Author:
Date:
2018-01-05
[Abstract]  Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017). [摘要]  在这里我们描述了两种测定方法来测量神经内分泌细胞中的致密核心囊泡(DCV)胞吐作用介导的货物分泌。 为了在受调控的DCV胞吐作用中对新基因进行siRNA筛选,我们开发了使用DCV货物,NPY-金星以及正交3H-血清素分泌测定的基于读板器的分泌测定法。 NPY-金星分泌测定成功地用于高通量siRNA筛选,并且使用血清素分泌测定来验证从筛选中鉴定的命中(Sorensen,2017; Zhang等人,2017)。


【背景】致密核心囊泡(DCV)胞吐作用介导内分泌和神经内分泌细胞分泌蛋白质,肽和小分子。当蛋白质和肽激素被内质网上的核糖体合成时,蛋白质和肽激素进入分泌途径,并在跨高尔基网络(TGN)(Borgonovo等人,2006; Bowman等, >等人,2009)。小分子物质,如5-羟色胺,直接被胞质单胺转运蛋白(VMATs)从细胞质直接吸收到DCV的内腔(Sudhof,2004)。在非刺激条件下,货物分子被储存在靠近质膜的DCV内。当细胞接受增加细胞质Ca ...

Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes
Author:
Date:
2017-12-20
[Abstract]  In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and ... [摘要]  在这篇论文中,我们描述了制备脑突触体的方案。突触体是用于分析突触的结构组分以及用于调查突触功能的有价值的模型系统。突触体制备对于理解可能发生突触蛋白的关键翻译后修饰的突触处的分子变化是必需的。突触小体不仅含有丰富的突触蛋白,还可用于分析神经递质向突触小泡的摄取和神经递质合成和释放的参与分析。可以用增加的钙内流刺激突触体释放神经递质。突触体制剂已被用于表征钙依赖性磷酸化和GABA合成酶GAD65(分子量为65kDa的L-谷氨酸脱羧酶)的活化。通过检查从突触体制剂获得的突触小泡膜上的蛋白质复合物,可以表征GAD65在GABA囊泡释放的偶联合成和囊泡摄取中的作用,这最终导致GABA囊泡释放GABA能神经传递的微调方法。

【背景】突触体制备方法在40多年前在神经科学研究实验室中建立,并且在涉及神经递质释放相关的细胞外钾升高以及对细胞内钙增加的应答方面具有极大的价值。除了阐明神经递质释放的过程之外,突触体制剂已经成为突触囊泡的有价值的来源。关于突触小泡的研究已经被用于表征参与偶联的神经递质合成和囊泡释放的蛋白质组分。突触体制剂作为突触囊泡分离的中间体也是非常有价值的,然后根据位于包含神经递质合成酶的囊泡膜上的蛋白质复合物进行分析。在这方面关键的意义在于包括CSP(胱氨酸 - ...

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