Summary

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Published: June 07, 2019
doi:

Summary

提出了一个程序,以可视化蛋白激酶A活动在头部固定,行为小鼠。改进的A-激酶活动报告器tAKAR®在皮质神经元中表达,并通过颅窗口进行成像。双光子荧光寿命成像显微镜用于在强制运动期间可视化体内的PKA活动。

Abstract

神经调节对大脑功能有强大的控制作用。神经调节系统功能障碍导致神经和精神疾病。尽管技术非常重要,但用细胞分辨率跟踪神经调节事件的技术才刚刚开始出现。神经调节剂,如多巴胺、去甲肾上腺素、乙酰胆碱和血清素,通过各自的G蛋白耦合受体触发细胞内信号事件,以调节神经元兴奋性、突触通信和其他神经元功能,从而调节神经元网络中的信息处理。上述神经调节器收敛到cAMP/蛋白激酶A(PKA)通路。因此,在体内,具有单细胞分辨率的PKA成像被开发为神经调节事件的读出,其方式类似于神经元电活动的钙成像。在此,提出一种方法,在头部固定行为小鼠皮层的各个神经元水平上可视化PKA活动。为此,使用了一个改进的A-激酶活动报告器(AKAR),称为tAKAR®,它基于Fürster共振能量转移(FRET)。这种基因编码的PKA传感器通过DNA质粒的子宫电穿孔(IUE)或腺相关病毒(AAV)的立体注射引入运动皮层。FRET 变化使用双光子荧光寿命成像显微镜 (2pFLIM) 进行成像,该显微镜比比例 FRET 测量具有优势,用于量化光散射脑组织中的 FRET 信号。为了研究强迫运动期间的PKA活动,tAKAR®通过清醒、头部固定的小鼠皮层上方的慢性颅窗成像,这些小鼠在速度控制的电动跑步机上运行或休息。这种成像方法将适用于许多其他大脑区域,以研究相应的行为诱导的PKA活动和其他基于FLIM的传感器进行体内成像。

Introduction

神经调节,也称为缓慢突触传输,在不同的行为状态(如压力、觉醒、注意力和运动1、2、3)期间对大脑功能施加强有力的控制。4.尽管其重要性,神经调节事件发生的时间和地点的研究仍处于起步阶段。神经调节剂,包括乙酰胆碱、多巴胺、肾上腺素、血清素和许多神经肽,激活G蛋白耦合受体(GPCRs),进而触发细胞内第二信使通路,时间范围很宽从秒到小时。虽然每个神经调节器触发一组不同的信号事件,cAMP/蛋白质激酶A(PKA)通路是许多神经调节器1,5的常见下游通路。cAMP/PKA通路调节神经元兴奋性、突触性传播和可塑性6,7,8,9,因此,调节神经元网络动力学。由于不同的神经元或神经元类型表达不同类型或水平的神经调节器受体10,同一细胞外神经调节器的细胞内效应可能是异质的,因此,必须研究与细胞分辨率。迄今为止,在行为过程中监测体内单个神经元的神经调节事件仍然具有挑战性。

为了研究神经调节的时空动力学,需要一种合适的记录方式。微透析和快速扫描循环伏特经常用于研究神经调节器的释放,但它们缺乏监测细胞事件11、12的空间分辨率。PKA成像与钙动力学一样,在群体成像13中用作神经元电活动的代理,PKA成像可用于以细胞分辨率读取神经元群中的神经调节事件。本协议描述了使用改进的A-激酶活动报告器(AKAR)来监测动物行为期间体内的PKA活动。此处描述的方法允许以亚细胞分辨率同时成像神经元群,其时间分辨率可跟踪生理神经调节事件。

AKAR由一个供体和接受荧光蛋白组成,由PKA磷酸化基质肽和叉头相关(FHA)域连接,与基质14、15的磷酸化丝氨酸或三甲氨酸结合。PKA通路激活后,AKAR的基质肽被磷酸化。因此,FHA 域与磷酸化基质肽结合,从而使两个荧光苷酸接近,称为 AKAR 的闭合状态。磷酸化AKAR的封闭状态导致供体和受体荧光量之间增加Fürster共振能量转移(FRET)。由于磷酸化ARNA的比例与PKA活性16的水平有关,生物样品中的FRET量可用于量化PKA活性16、17、18的含量。 19,20.

早期版本的AKARs主要设计为双色比例成像14。当成像深入到脑组织,比例法遭受信号失真,由于波长依赖光散射17,18,21。如下所述,荧光寿命成像显微镜(FLIM)消除了这个问题,因为FLIM只测量供体荧光量18、21发出的光子。因此,FRET 的 FLIM 定量不受组织深度17的影响。此外,可以使用接受器荧光的”暗”(即低量子收率[QY])变体。这释放了一个颜色通道,以方便通过同步成像第二个传感器或形态标记17,19,20的正交神经元属性的多路测量。

FLIM成像量化荧光在兴奋状态下花费的时间,即荧光寿命18。荧光回到地面状态,从而结束兴奋状态,通常伴随光子的发射。虽然单个兴奋分子的光子发射是随机的,但在人群中,平均荧光寿命是该特定荧光的特性。当纯荧光群同时激发时,产生的荧光将跟随单个指数衰变。此指数衰减的时间常数对应于荧光蛋白的平均荧光寿命,荧光蛋白通常为 1 到 4 纳秒。FRET 也可以将兴奋的供体荧光剂返回到地面状态。在FRET的存在下,供体荧光的荧光寿命缩短。非磷酸化的AVR表现出相对较长的供体荧光寿命。在PKA的磷酸化后,传感器的寿命较短,因为供体和受体的荧光量彼此靠近,FRET增加。因此,在AKARs的种群中荧光寿命的量化代表PKA活性水平。

早期版本的AKARs尚未成功用于单细胞分辨率的体内成像。这主要是由于AKAR传感器对生理活化信号振幅低。最近,通过系统地比较两光子荧光寿命成像显微镜(2pFLIM)的可用AKAR传感器,发现一种名为FLIM-AKAR的传感器性能优于替代传感器。此外,还开发了一系列称为靶向AKARs(tAKARs)的FLIM-AKAR变体,以可视化特定亚细胞位置的PKA活性:微管(tAKAR®)、细胞醇(tAKAR®)、活性素(tAKAR®)、丝状活性素(tAKAR®)、膜(tAKARé)、膜(tAKARé)、微管(tAKAR®)、细胞醇(tAKAR®)、活性素(tAKAR®)、膜(tAKARé)、膜(tAKARé)、微管(tAKAR®)、细胞源性活性素(tAKAR®)、活性剂(tAKAR®)。(tAKAR®),和鼻后密度(tAKAR*)。在tAKARs中,tAKAR®将去甲肾上腺素引起的信号振幅提高了2.7倍。这与神经元中的大多数PKA固定在静止状态22、23的微管上的知识是一致的。tAKAR® 是现有 2pFLIM 的 AVR 中表现最好的。此外,tAKAR®检测到由多个神经调节剂引起的与生理相关的PKA活性,tAKAR®的表达并没有改变神经元功能17。

最近,tAKAR®成功地用于可视化头固定行为小鼠17中的PKA活动。结果表明,强制运动触发了运动、桶和视觉皮质中表层神经元(第1层至3层,距离pia深度为+300μm)的PA活性。运动触发的PKA活性部分依赖于通过β-肾上腺素受体和D1多巴胺受体的信号,但不受D2多巴胺受体拮抗剂的影响。这项工作说明了tAARs使用2pFLIM跟踪体内神经调节事件的能力。

在当前协议中,在强制运动范式期间,在头部固定的醒小鼠中进行 PKA 活动成像的整个方法用六个步骤来描述。首先,在传统的双光子显微镜中增加2pFLIM功能(图1)。第二,建造电动跑步机(图2)。第三,通过子宫电穿孔(IUE)的DNA质粒,或对腺相关病毒(AAV)的立体注射,在小鼠皮层中表达tAKAR®传感器。IUE24、25和立体注射病毒颗粒26的出色手术方案已经发表。我们使用的关键参数如下所述。第四,安装一个颅窗。优秀的方案已经发表颅窗手术27,28。介绍了从标准协议修改的几个步骤。第五,在体内表演2pFLIM。第六,分析2pFLIM图像(图3图4)。这种方法应该很容易适用于许多其他头部固定的行为范式和大脑区域。

Protocol

这里描述的所有方法都已获得俄勒冈健康与科学大学的机构动物护理和使用委员会(IACUC)的批准。 1. 2pFLIM 显微镜设置 安装光子定时计数模块(PTCM,材料表),并根据制造商手册连接到计算机(图1)。注:PTCM 通常是接收激光脉冲计时的”同步”输入和来自光电倍增管 (PMT) 的光子输入的计算机板。它还从双光子成像控制软件接收像…

Representative Results

FRET-FLIM 传感器允许可视化许多不同的信号通路,包括神经调节中涉及的 cAMP/PKA 通路。当前协议利用最近开发的 takaR® 传感器与 2pFLIM 相结合,在头部固定行为小鼠中可视化 PKA 活动。大多数现有的双光子显微镜可以通过添加三到四个组件来升级,如图 1所示(另请参阅第 1 节)。为了在2pFLIM采集的图像中可视化FRET,对每像素收集的光子计时的直方图图进行了?…

Discussion

该协议演示了使用 FRET-FLIM 传感器 tAKAR® 来可视化头部固定行为小鼠的神经调节触发的 PKA 活动。此应用基于 TAKAR® 体外和体内的广泛测试和表征,以证明获得的 FLIM 信号与生理神经调节事件17相关。在这里,一个在体内的应用,运动诱导的PKA活动在运动皮层,用于描述程序,提供传感器到大脑,动物手术成像,硬件和软件要求的行为和成像数据采集,用于成像数据分析的软件和算法。

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Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢苔丝·拉迈耶女士、露丝·弗兰克女士和迈克尔·穆尼亚克博士的编辑和评论,以及佛罗里达州马克斯·普朗克的Ryohei Yasuda博士对2pFLIM采集软件的介绍。这项工作得到了两个BRAIN倡议奖U01NS094247(H.Z.和T.M.)和R01NS104944(H.Z.和T.M.)的支持,一个R01授予R01NS081071(T.M.),以及R21授予R21NS097856(H.Z.)。所有奖项均来自美国国家神经疾病和中风研究所。

Materials

0.2 μm cellulose acetate syringe filter Nalgene 190-2520 Step 3.2.2.
16x 0.8 NA water-immersion objective Nikon MRP07220 Step 5.5.
3-pin cable US digital CA-MIC3-SH-NC Step 2.5. To connect rotation sensor to the DAQ input of the microscope
Aluminum bread board Thorlabs MB1012 Step 2.5.
AnimalTracker MATLAB software N/A N/A Step 2.5 and sections 5 – 6. Will be provided upon request to the lead author
Band-pass barrier filter Chroma ET500-40m Step 1.4.
Cage plate Thorlabs CP01 Step 2.4. Used as mount for rotation sensor
Carbon steel burrs for micro drill, 0.5 mm tip diameter FST 19007-05 Steps 3.2.3. and 4.4.
Circular coverslip (5mm diameter) VWR 101413-528 Step 4.5.
Custom-made injection needle holder N/A N/A Step 3.2.4. Technical details provided upon request to the lead author
Dental acrylic Yates Motloid 44114 Steps 4.3. and 4.5.
Dental drill; Microtorque ii Ram products 66699 Steps 3.2.3. and 4.4.
Dowsil transparent polymer The Dow Chemical Company 3-4680 Step 4.5. Artificial dura
Electroporation electrode Bex LF650P5 Step 3.1.4.
Electroporator Bex CUY21 Step 3.1.4.
Fast green FCF Sigma-aldrich F7258-25G Step 3.1.1.
FLIMimage MATLAB software N/A N/A Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida
FLIMview MATLAB software N/A N/A Sections 5. and 6. Will be provided upon request to the lead author
Foam-compatible glue (Gorilla White Glue) Gorilla 5201204 Step 2.3.
Headplate N/A N/A Step 4.3. Technical details provided upon request to the lead author
Headplate holder N/A N/A Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket
Hydraulic micromanipulator Narishige MO-10 Step 3.2.4.
Krazy glue Krazy glue KG82648R Step 4.3. Cyanoacrylate-based glue
Low-noise fast photomultiplier tube Hamamatsu H7422PA-40 or H10769PA-40 Step 1.3.
MATLAB 2012b Mathworks N/A Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software
Motor Zhengke ZGA37RG Step 2.4.
Motor speed controller Elenker EK-G00015A1-1 Step 2.5.
Motorized micromanipulator Sutter MP-285 Step 3.2.4.
Mounting base Thorlabs BA1S Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2
Mounting post Thorlabs P14 Step 2.6. Used for headplate holder post in combination with PB2
Mounting post base Thorlabs PB2 Step 2.6. Used for headplate holder post in combination with P14
Mounting post bracket Thorlabs C1515 Step 2.6. Used in combination with right-angle bracket and headplate holder
Optical post Thorlabs TR2 Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4
Phosphate-buffered saline Ν/Α Ν/Α Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247
Photodiode Thorlabs FDS010 Step 1.2.
Photon timing counting module Becker and Hickl SPC-150 Step 1.1.
Plasmid: tAKARα (CAG-tAKARα-WPRE) Addgene 119913 Step 3.1.3.
Post holder Thorlabs PH4 Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2
Right-angle bracket Thorlabs AB90 Step 2.6 Used in combination with mounting post bracket and headplate holder
Rotation sensor US digital MA3-A10-250-N Step 2.4.
Rubber mat Rubber-Cal B01DCR5LUG Step 2.1.
Shaft coupling (1/4 inch x 1/4 inch) McMaster 6208K433 Steps 2.3. and 2.4.
ScanImage 3.6 Svoboda Lab/Vidrio Technology N/A Steps 5.9. and 6.1.
Signal splitter Becker and Hickl HPM-CON-02 Step 1.3.1.
Stainless steel axle (diameter 1/4 inch, L = 12 inch) McMaster 1327K66 Step 2.3.
Stereotaxic alignment systsem David kopf 1900 Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator
Two-photon microscope N/A N/A Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)
Vetbond tissue adhesive 3M 14006 Step 3.2.6.
Virus: tAKARα (AAV2/1 hSyn-tAKARα-WPRE) Addgene 119921 Step 3.2.2.
White PE foam roller (8 x 12 inch) Fabrication enterprises INC. 30-2261 Step 2.1.1.
White polystyrene fom ball halves GrahamSweet 200mm diameter 2 hollow halves Step 2.1.1.
Zipkicker PACER PT29 Step 4.3. Hardening accelerator

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Cite This Article
Jongbloets, B. C., Ma, L., Mao, T., Zhong, H. Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy. J. Vis. Exp. (148), e59526, doi:10.3791/59526 (2019).

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