在黑质和腹侧被盖区多巴胺调控的综合性能分析

Summary

多巴胺是明显的调节中脑核，其中包含的多巴胺神经元的胞体和树突。在这里，我们描述了一次解剖和样品处理方法效果发挥到极致，因此，结论和见解，在黑质（SN）和腹侧被盖区（VTA）在啮齿动物的脑核的多巴胺调节。

Abstract

多巴胺是一种中枢神经系统的大力研究的神经递质。事实上，在自发活动的参与和奖励有关的行为，促进多巴胺调控的分子缺陷五十年的调查。纹状体和伏隔核的多巴胺调控时，大脑的分子基础，其在终端领域的黑质纹状体和mesoaccumbens途径地区的监管重点在这些查询大多数。此外，这些研究都集中在多巴胺的组织内容的分析与正常化的唯一的湿纸巾重量。调查的蛋白质，调节多巴胺，如酪氨酸羟化酶（TH）蛋白，TH磷酸化，多巴胺转运体（DAT），2囊泡单胺转运蛋白（VMAT2），通常不包括多巴胺的组织内容在同一样品的分析。同时分析多巴胺的组织内容及其调节蛋白（包括后TRA的能力nslational修改），不仅提供了内在的力量来解释多巴胺与TH，DAT或VMAT2蛋白水平和功能的关系，而且还扩展了样本经济。这意味着到更低的成本，但会产生多巴胺的分子调控，在几乎所有的研究者选择的范式的见解。

我们的分析集中在中脑。虽然最多巴胺调控的研究通常被忽视的SN和腹侧被盖区，这些原子核很容易解剖与实践。可以进行一个全面的读数，多巴胺的组织内容和TH的DAT，或VMAT2。行为在SN和腹侧被盖区的多巴胺功能的影响有正蓬勃发展的文学，和外源性物质或疾病的进程，其中^1-5 impingements。此外，如生长因子的化合物对多巴胺和多巴胺调节蛋白产生深远的影响，到相对更大程度上的SN或腹侧被盖区<sup> 6-8。因此，这种方法提出参考实验室，希望延长其具体的治疗方法如何调节行为和多巴胺调控的查询。在这里，一个多步骤的方法提出了多巴胺的组织内容的分析，的TH蛋白水平的DAT或VMAT2，啮齿动物脑黑质和腹侧被盖区的磷酸化和TH。 TH的磷酸化分析，不仅是如何调节TH活力，而且还影响级联信号在一个给定的范例somatodendritic核可以产生显着的见解。

我们将说明夹层技术来分隔这两个核和解剖组织来样加工，产生的文件，揭示在体内的多巴胺调控的分子机制，具体的每一个细胞核（图1）。

Protocol

1。解剖在湿冰床，放松啮齿类动物的大脑基质（冠状切片分离1 mm处），培养皿中含有5剃须刀，＃11手术刀。在一个单独的容器中，地方标记成干冰2毫升大小的离心管。 研究者会选择安乐死的方法。我们有可重复性的结果，理想的情况下，异氟醚蒸发器应该被用来进行非常简短的麻醉。但是，如果不提供，我们使用一个大型的电瓶包含一个平台。在位于批准通风罩电瓶，将异氟醚?…

Discussion

正如图1中所述，上面详细介绍的方法应该产生多巴胺和调节蛋白TH，的DAT，从一个样品SN或腹侧被盖区VMAT2的大鼠或小鼠获得多个读数。再次，开展这项协议的好处是，研究人员可以得到多巴胺是如何调节体内几乎所有的实验范式下，这样做，保存在任何所需的动物数量减少的重要实验资源的业务相匹配的读数实验。

这是绝对必要的，研究者观察在夹层（4℃），存储组…

Materials

HPLC system:

The basic system consists of a Shimadzu LC10-ADvp HPLC pump, a Waters WISP 717 automatic sample injector, a 250 X 4.4 mm 5 micron Spherosorb ODS-1 C18 reverse-phase column (Waters), a Bioanalytical Systems (BAS) TL12 dual glassy carbon electrode, two BAS LC4B electrochemical detectors, and a Waters Empower 2 data collection and integration system.

The column is maintained at 30-45°C (BAS LC22A column heater). The mobile phase is 0.1 M sodium phosphate (pH 3.0), 0.1 mM EDTA, 0.2-0.4 mM 1-Octane Sulfonic Acid (Eastman-Kodak), and 0.35% acetonitrile (v/v), filtered through a 0.45 micron filter. Flow rate is of 1.2 ml/min. Four liter batches of mobile phase are optimized for separations by adjusting the pH, Octane Sulfonic Acid and column temperature. The mobile phase is recycled, and is continuously purged with helium gas to remove dissolved oxygen. Recycling of the mobile phase is almost essential to maintain good resolution for a reasonable period of time. The mobile phase shelf-life is maintained by using a flow switch (controlled by the integrator) to divert to waste the first 2-7 min of each run.

The electrodes are maintained at potentials of approximately 0.78 and 0.95V with respect to a Ag/AgC1 reference electrode. The electrode at the higher potential is used exclusively for the determination of tryptophan (and the NMDA internal standard). The 0.78 V potential provides a superior signal to noise ratio for detection of the monoamines and compounds, other than tryptophan. The chromatograms are stored on the hard drive of the Empower workstation, and subsequently processed and the data transferred directly into an Excel spreadsheet for computation of metabolite amounts and compilation of group data.

Pump: Shimadzu LC-10AD
Cell: BAS Cross Flow. Glassy carbon working electrode at 0.780 and 0.950 V potential.
Detector: BAS LC-4B operated in dual channel mode.
Data Acq. System: Waters Empower Pro 2.
Injector: Waters WISP 717
Column: Waters Spherosorb ODS-1, 5 μM particle, 4.4 mm X 250 mm.

Name of the reagent	Company	Catalogue number
Sodium Dodecyl Sulfate (SDS)	– J.T. Baker	4095-02
Trizma Base	Sigma	T1503-1KG
Trizma HCl	Sigma	T3253-1KG
Glycerol	Sigma	G8773-500 mL
PVP-40	Sigma	PVP40-1KG
dPBS	Gibco	21600-069
Tween20	Sigma	P1379-500 mL
Glycine	Sigma	G8898-1KG
Ponceau S	Fluka	81460
Bromophenol Blue	Sigma	B8026-5G
Dithiothreitol	Sigma	D-9163
Protein Standard 2 mg BSA	Sigma	P5619-25VL
Pierce BCA Protein Assay Reagent A	Thermo- Fisher Scientific	23223
Precision Plus Protein Standard	Bio Rad	161-0373
[125I]-protein A, specific activity	Perkin-Elmer

Table 2. Specific reagents.

Reagents	Formulas
10% SDS	10 g SDS, 100 mL DI H20
1% SDS (pH to 8.2)	10 mL 10%SDS 60.5 mg Trizma Base 37.22 mg EDTA 90 mL DI H20
Copper II Sulfate Solution	1 g Copper II sulfate 25 mL DI H20
3X Sample Buffer	Trizma Base 2.27 g SDS 6 g Dithiothreitol 0.463 g Glycerol 30 g Bromophenol Blue 10 mg D I H20 (initially add above reagents to 40 mL of H20 in a graduated cylinder; then add H20 until volume reaches 100 mL) HCl (add as needed to reach pH of 6.85) Freeze solution in 50 2.0 mL tubes. (makes 100 mL): Volume of 3X Sample Buffer needed = ½ volume of SDS used in sample.
1X Sample Buffer	Dilute 3X Sample Buffer down to 1X sample buffer using DI H20
10X Running Buffer (Makes 4 L)	Trizma Base 121.1 g Glycine 577 g SDS 40 g
10X Transfer Buffer: (Makes 4 L)	Glycine 360 g Trizma Base 96 g
Ponceau	Ponceau S. 0.5 g Acetic Acid 5 mL DI H20 95 mL
.2% HCl Solution	5.2 mL HCl in 500mL of DI H20
PVP-T20 Blocking Soln. (Makes 4 L)	PVP-40 40 g dPBS 38.2 g Tween20 2 g Thimerisol 0.4 g 1M Tris pH 7.6 (60.6 g Tris HCl + 13.9 g Tris Base in 500 mL DI H20)- 50 mL
10X Blot Buffer (Makes 4 L)	Tween 20 20 g Tris Base 14 g Tris HCl 61 g

Table 3. Protein Assay and Western Blotting Formulas.

Tyrosine hydroxylase standards: The calibrated TH protein and phosphorylation standards used by this laboratory are derived from PC12 cell extracts, which were analyzed for TH protein content and phosphorylation stoichiometries against a previously calibrated TH standards that ultimately originated from the laboratory of Dr. John Haycock ¹¹.