定量自动放射法测定维沃脑蛋白合成区域速率
Summary
蛋白质合成是细胞的关键生物过程。在大脑中,它是自适应变化所必需的。测量完整大脑中蛋白质合成率需要仔细的方法学考虑。这里我们介绍了L-[1-14C]-亮细胞定量自射方法,用于测定体内脑蛋白合成的区域速率。
Abstract
蛋白质合成是神经元功能发育和维护所必需的,并参与神经系统的适应性变化。此外,人们认为神经系统蛋白质合成障碍可能是某些发育障碍的核心表型。准确测量动物模型中脑蛋白合成率对于了解这些疾病非常重要。我们开发的方法被设计用于研究醒着、有行为的动物。它是一种定量的自动放射学方法,因此可以同时产生大脑所有区域的速率。该方法基于示踪氨基酸L-1-14 C+-亮氨酸的使用,以及L-亮氨酸在大脑中行为的动力学模型。我们选择L-1-14 C+-亮氨酸作为示踪剂,因为它不会导致外在标记的代谢产物。它要么被整合到蛋白质中,要么被快速代谢,产生14CO2,在大脑中大量未标记的CO2中稀释。 该方法和模型还允许从组织蛋白解基中提取的无标记亮氨酸对蛋白质合成的组织前体池的贡献。该方法具有空间分辨率,可确定细胞和神经层以及下丘脑和颅神经核的蛋白质合成率。为了获得可靠且可重复的定量数据,必须遵循程序细节。这里我们介绍了定量自放射学L-1-14 C+-亮氨酸法的详细程序,用于确定体内蛋白质合成的区域速率。
Introduction
蛋白质合成是神经系统长期适应性变化的重要生物过程。抑制蛋白质合成会阻止无脊椎动物和脊椎动物的长期记忆储存2。蛋白质合成对于维持某些形式的长期强效 (LTP) 和长期抑郁症 (LTD) 3、发育过程中的神经元存活率4以及神经元及其一般维持的后期阶段至关重要突触连接5.大脑蛋白质合成率的测量可能是研究适应性变化以及与学习和记忆相关的神经发育障碍和紊乱的重要工具。
我们已经开发出一种量化清醒动物体内脑蛋白合成率的方法,它与其他估计脑组织外体或体外制剂速率的技术具有固有的优势。最重要的是适用于清醒动物大脑中完整大脑的测量。这是一个关键的考虑因素,因为它允许具有突触结构和功能的测量,而不必担心验尸后的效果。此外,我们采用的定量自动放射学方法实现了高度的空间定位。虽然14C的能量是,我们不能本地化示踪剂在亚细胞或细胞水平,我们可以测量速率的细胞层和小大脑区域,如下丘脑核,大约25μm分辨率7。
用放射性追踪器进行体内测量的一个挑战是确保测量的放射性标签是在感兴趣的反应产物中,而不是未反应的标记前体或其他无关标记的代谢产物6。我们选择L-1-14 C+-亮氨酸作为示踪氨基酸,因为它要么被整合到蛋白质中,要么迅速代谢成14CO2,在大脑中未标记的CO2大池中稀释,因为高速率能量代谢8.此外,任何14C未纳入蛋白质主要存在作为自由[14 C]-亮氨酸,在60分钟的实验期间,几乎完全从组织6清除。蛋白质然后固定到组织与形式素,然后用水冲洗,以去除任何免费的 [14C]-亮氨酸之前,自体。
另一个重要考虑因素是组织蛋白解中未标记氨基酸稀释前体氨基酸池特定活性的问题。我们已经表明,在成年大鼠和小鼠中,大脑中蛋白质合成的前体亮氨酸池中约有40%来自来自蛋白质分解6的氨基酸。这必须包括在脑蛋白合成(rCPS)的区域速率的计算中,并且必须在可能改变这种关系的研究中得到确认。该方法的理论基础和假设已在其他地方详细介绍。本文着重论述了该方法应用的程序问题。
该方法已用于测定地松鼠9,绵羊10,河河猴11,大鼠12,13,14,15,16的rCPS,17,18,19,20,21、一种小鼠模型的管状硬化复合22,小鼠模型脆弱的X综合征23,24,25,26,脆弱的X预突变小鼠27,和苯丙酮尿的小鼠模型28。在本手稿中,我们介绍了使用体内自放射学 L-1-14 C+-亮氨酸法测量rCPS 的程序。我们在醒着的控制鼠标的大脑区域中呈现rCPS。我们还证明,在体内对一种翻译抑制剂的抗声霉素的分管,可以消除大脑中的蛋白质合成。
Protocol
注:所有动物程序均获得国家精神卫生动物护理和使用委员会的批准,并按照国家卫生研究院《动物护理和使用指南》进行。 图 1中提供了该协议的概述。 1. 分别在股骨静脉和动脉中植入导管,分别用于给导管和采集时导动脉血样。在给动剂前至少22小时完成手术。手术大约需要1小时才能完成。 收集必要的材料:无菌手术器械(手…
Representative Results
这里展示了一个代表性的实验,演示了蛋白质合成抑制剂先前对rCPS的影响。正常盐水中的苯二恶客在开始rCPS测定前30分钟给一只成年C57/BL6雄性野生型小鼠皮下(100毫克/千克)施用。与车辆处理的对照动物相比,异种素治疗的效果表明,在异种霉素处理小鼠中,rCPS几乎无法检测到(图4)。这些数据表明,体内自放射学L-1-14 C+-亮氨酸法测量大脑中蛋白质?…
Discussion
提出了一种定量测定实验动物体内脑蛋白合成(rCPS)区域速率的方法。与现有方法的方法,这种方法具有相当大的优势:1.测量是在清醒的有行为动物中进行的,因此它们反映了大脑中正在进行的过程。2. 测量是通过定量自辐射法进行的,能够同时确定大脑所有区域和分区域的rCPS。3. 该方法的动力学模型考虑到从组织蛋白降解中产生的未标记氨基酸的回收可能性及其对蛋白质合成前体池的影响6。<sup class=…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢曾燕夏对小鼠的基因分型,汤姆·伯林的氨基酸和薄膜的加工,和梅琴进行一些rCPS实验。这项研究得到了NIMH的内学研究计划,ZIA MH00889的支持。RMS 还得到了自闭症讲博士后奖学金 8679 和 FRAXA 博士后奖学金的支持。
Materials
| Mice | The Jackson Laboratory | 003024 | Fmr1 knockout breeding pairs |
| Anisomycin | Tocris Bioscience | 1290 | |
| Microhematocrit Tubes | Drummond Scientific | 1-000-3200-H | capillary tubes |
| Critoseal Capillary Tube Sealant | Leica Microsystems | 39215003 | sealant putty |
| Glass vial inserts | Agilent | 5183-2089 | used to collect blood samples |
| Digi-Med Blood Pressure Analyzer | Micro-Med Inc. | BPA-400 | blood pressure analyzer |
| Bayer Breeze 2 Blood Glucose Monitoring System | Bayer Breeze | 9570A | glucose meter |
| Gastight syringe | Hamilton Co. | 1710 | tuberculin glass syringe |
| HeatMax HotHands-2 Hand Warmers | HeatMax | Model HH2 | warming pads |
| Heparin Lock Flush Solution | Fresenius Kabi USA, LLC | 504505 | heparin saline |
| Clear animal container | Instech | MTANK/W | animal enclosure |
| Spring tether | Instech | PS62 | catheter tube/rodent attachment |
| Swivel | Instech | 375/25 | hooks to spring tether |
| Swivel arm and mount | Instech | SMCLA | hooks to swivel and animal enclosure |
| Tether button | Instech | VAB62BS/22 | attaches to bottom of spring tether |
| Stainless steel tube | Made in-house | N/A | used to snake catheters through mouse |
| Matrx VIP 3000 | Matrx | 91305430 | isoflurane vaporizer |
| Isoflurane | Stoelting Co. | 50207 | isoflurane/halothane adsorber |
| Clippers | Oster Finisher | Model 59 | |
| Surgical skin hooks | Made in-house (??) | N/A (??) | |
| 0.9% Sodium Chloride Saline | APP Pharmaceuticals LLC | 918610 | |
| Forceps | Fine Science Tools | 11274-20 | |
| Surgical scissors | Fine Science Tools | 14058-11 | |
| Microscissors | Fine Science Tools | 15000-00 | |
| UNIFY silk surgical sutures | AD Surgical | #S-S618R13 | 6-0 USP, non-absorbable |
| PE-8 polyethylene tubing | SAI Infusion Technologies | PE-8-25 | |
| Syringe | Becton Dickinson and Co. | 309659 | 1cc/mL |
| PE-10 polyethylene tubing | Clay Adams | 427400 | |
| MCID Analysis | Imaging Research Inc. | Version 7.0 | optical density analysis |
| Gelatin-coated slides (75x25mm) | FD Neurotechnologies | PO101 | |
| Cryostat | Leica | CM1850 | |
| Super RX-N medical x-ray film | Fuji | 47410-19291 | |
| Hypercassettes (8×10 in) | Amersham Pharmacia Biotech | 11649 | |
| [1-14C]leucine | Moravek | MC404E | |
| Microcentrifuge tube | Sarstedt Aktiengesellschaft & Co. | 72.692.005 | used to deproteinize blood samples |
| Glass pasteur pipette | Wheaton | 357335 | |
| Glass wool | Sigma-Aldrich | 18421 | |
| Nitrogen | NIH Supply Center | 6830009737285 | |
| Scintillation fluid | CytoScint | 882453 | |
| Liquid scintilllation counter | Packard Tri-Carb | 2250CA | |
| Amino acid analyzer | Pickering Laboratories | Pinnacle PCX | |
| HPLC unit | Agilent Technologies | 1260 Infinity | include 1260 Bio-Inert Pump |
| Surgical microscope | Wild Heerbrugg | M650 | |
| Sulfosalicylic acid | Sigma-Aldrich | MKBS1634V | 5-sulfosalicylic acid dihydrate |
| Norleucine | Sigma | N8513 | |
| 1.0 N HCl | Sigma-Aldrich | H9892 | |
| [H3]leucine | Moraevk | MC672 | |
| Falcon tube | Thermo Scientific | 339652 | 50 mL conical centrifuge tubes |
| Stopwatch | Heuer Microsplit | Model 1000 | 1/100 min |
| Euthanasia Solution | Vet One | H6438 | |
| Northern Light Precision Illuminator | Imaging Research Inc. | Model B95 | fluorescent light box |
| Micro-NIKKOR 55mm f/2.8 | Nikon | 1442 | CDD camera |
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Citar este artigo
Saré, R. M., Torossian, A., Rosenheck, M., Huang, T., Beebe Smith, C. Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo. J. Vis. Exp. (148), e58503, doi:10.3791/58503 (2019).