Summary

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

Published: June 22, 2015
doi:

Summary

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

Abstract

这个协议描述如何暂时和远程沉默神经元活动中的离散的大脑区域,而动物从事学习和记忆任务。该方法结合了药物遗传学(设计 – 受体 – 独占激活的逐设计-用药)与行为范式(感官预处理),其被设计不同形式的学习的区别开来。具体地,病毒介导的递送是用来表达基因修饰抑制G-蛋白偶联受体(设计器受体)插入在啮齿动物的离散大脑区域。三周后,当设计师受体表达水平是高的,药理剂(在设计药物)的全身给药30分钟之前,一个特定的行为会话。该药具有设计师受体的亲和力,从而导致抑制表达设计师受体神经元,但在其他生物惰性。该大脑区域保持沉默2-5小时(depen鼎上的剂量和给药途径)。在行为模式完成后,脑组织被评估正确的位置和受体的表达。这种方法是用于确定个体的大脑区域,以行为的特定组件的贡献,并且可以跨越任何数量的行为范例的使用是特别有用的。

Introduction

行为神经科学领域内一个令人兴奋的挑战是确定复杂行为的神经基础。许多如永久性病变,通过套管植入大脑暂时失活和光遗传学技术已经被用来确定离散的大脑区域的贡献了复杂行为的子组件。虽然在学习这些方法告知我们的区域特殊性的理解,每种技术也不是没有限制的。具体地讲,永久病灶通常进行前行为测试,因此它们的作用是在整个范例的持续时间存在。插管研究涉及一个短期神经灭活剂( 例如,河豚毒素)的呈现可以产生于脑组织实质损害,并且可以刚好在行为测试诱导应力的受试者。此外,通过插管灭活限于组织围绕的区域尖端的插管。最后,虽然光遗传学提供了一系列的灵活性可满足特定的大脑区域活动的时间控制,它是成本过高,技术要求高。

这些限制可以使用药理学方法(设计-受体独占激活的按设计药物,DREADDs)1,2有待克服。重要的是,而药物基因学的概念是复杂的,该技术的执行是简单的。类似于涉及输液毒素( 例如,NMDA,鹅膏蕈氨酸)转换成离散的大脑区域,这种技术涉及输注腺相关病毒(AAV),其中包含的DNA片段进行修饰抑制G蛋白偶联型受体的传统立体手术方法(hM4Di;设计者受体)进入的标准实验室啮齿类感兴趣的区域( 见图1)。所述病毒载体还含有荧光报道(mcitrine)。一旦合并到细胞中,设计者受体(和报告蛋白)的最大限度表达〜后3周输注和2-5小时由否则生物学惰性设计者药物的全身给药,氯氮平-N-氧化物可以被选择性地激活(CNO)1 3。因为实验者被赋予了超过在特定脑区域的神经活动的精确的,但遥控临时控制,药物遗传学结合特别好地与在多个阶段进行的行为范式。在这个例子中,retrosplenial皮质(RSC)的贡献对刺激刺激学习相比,其在巴甫洛夫学习的作用,然而办法这种组合非常适合于设法确定脑区如何具体向任意数量的问题复杂的行为。

此外,虽然在本协议中没有描述,病毒和转基因的方法可以用来实现细胞类型特异性DREADD表达式2。正如我固有在涉及药理学和/或其它类型的实验操作,仔细考虑的实验设计和随后的定量分析的行为范例是采用DREADD方法时需要。实验者新来的DREADD方法被称为当前DREADD 2技术的全面审查。

每一天,生物体了解新的刺激和事件以及它们彼此之间的关系。甚至在熟悉的环境,如家庭,一种是快速的以检测更改在刺激之间的关系,因为这些变化可能是预测有意义的事件。这种刺激的刺激( 即,关系)学习涉及多种刺激的互联以及历来与海马,其中心位于内侧颞叶4内相关联。但是,海马并不存在,也没有采取行动,孤立;内和出皮层区域颞叶内侧的一边提供关键的感官信息的海马结构5-7。传统的永久损伤研究提供了令人信服的证据的一个数字皮层区域( 例如,retrosplenial,postrhinal和内嗅皮质)中的海马依赖性学习的参与,但在其过程中的离散的阶段辨别一个特定区域的作用的能力是有限的学习8-10。

本协议测试的假设,即在RSC是必要的刺激-刺激的学习期间通过一个3相感官预处理范例11,12的单相沉默在RSC。简而言之,大鼠收到包含设计者受体和〜3周后施用设计者药物(CNO)30前行为测试开始分钟的AAV的输注。在本协议中,实验组大鼠在测试过程中的第一阶段(当刺激刺激李尔收到CNO宁发生),他们在接下来的2阶段测试接收车辆。为了控制CNO对行为的影响意外,注入老鼠与设计师受体(hM4Di),并注入一些车辆,而不是CNO。考虑到病毒输注和受体表达的一般效果,注入一个对照病毒不包含设计者受体和管理CNO。

许多AAV的不同血清型的用于输送遗传物质。研究涉及重组或合成的分子的当前的NIH指导方针主张的AAV(所有血清型)和重组的或合成的AAV构建体,其中所述转基因不编码任一种潜在的致瘤基因产物或毒素分子,并且产生在没有一辅助病毒,需要BSL-1的预防措施(附录B-1。风险组1(RG1)代理)13。可用14,15一些有关AAV结构,实用性和安全审查的。值得注意的是,虽然时,由于有关在啮齿动物可能生殖16,17和潜在的致癌机制18-20关注,一些机构要求使用的BSL-2的预防措施的AAV工作时。验证通过与地方研究会进行,疾病控制中心和美国国立卫生研究院准则研究使用病毒载体的基因操作在美国时涉及重组DNA分子13的监督委员会,在个别机构咨询,使用前适当的BSL。个人防护,调查培训,矢量遏制,净化,消毒处理的材料,以及注射后的动物住房需求是由这些原则规定。此外,参阅并遵循适当的机构动物护理和使用委员会的准则或同等机构的监督委员会的指导方针,以确保安全处理,管理和处置的AAV。

Protocol

利用动物的奥伯林学院机构动物护理批准和使用委员会,并按照实验动物21与指南护理和使用。 1.准备输液病毒注:该协议使用BSL-1的预防措施。当采用BSL-2的预防措施,一次性白大褂,手套,鞋套,保护眼睛和防护口罩(N95型)是必需的。所有个人处理BSL-2化合物必须符合由当地公共卫生机构测试的微粒呼吸器。请参阅洛厄&Majewska(2010)22</su…

Representative Results

行为结果在实验结束后,将区域专用临时灭活的有效性应该被定量和定性评估。本实施例涉及一个3相行为范式(感官预处理),其中CNO施用以减弱在预处理会话中在RSC神经活动来检验这一假设在RSC是必要的协会之间中性刺激12的形成。重要的是,实验者不局限于行为范式或本文描述的药物遗传学方法可以加上最多行为范例实验设计。 而分析通常不?…

Discussion

这个协议描述了如何应用药理学方法(DREADD)调查如何特定脑区域有助于多相复杂的学习任务。随着跨学习阶段暂时和远程沉默的神经活动的离散的大脑区域的能力,这种方法相结合提供了一个平台,调查范围广泛的行为,包括学习更细致或屏蔽的形式。在这个协议中所描述的例子中,控制该表达在retrosplenial皮层(RSC)的设计者受体鼠和大鼠中的3相行为的任务12进行测试。任务的第一阶?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢罗宾逊等人 12的作者为他们从本议定书部分派生的手稿贡献。

Materials

Male, Long Evans Rats, 55-60 d Hilltop Lab Animals Inc
rAAV8/hSyn-HA-hM4D(Gi)-IRES-mCitrine Virus Vector Core Caution:  This is a BSL-1 compound
rAAV8/hSyn-GFP Virus Vector Core Caution:  This is a BSL-1 compound
Clozapine-N-Oxide R&D Systems 4936-10 Designer Drug
Rat Cage lid (Polycarbonate) Alternative Design  FT 8XL-PC Used to cover animal cages 48-72 hours post infusion
Filer Paper (Replacement) Alternative Design  FP-R-1018XAD Filter paper that goes with cage lids
Table Top Vise JETS 2201-265 For holding microscentrifuge tubes containing AAV in the hood
Medline Biohazard liners  Staples 113444 Biohazard Trash Bags
United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle  Amazon.com Biohazard Trash Can
Isoflurane, 100 mL Patterson Veterinary Supply Inc.  07-890-8540 Anesthetic
Dual Small Animal Stereotaxic with Digital Display Readout Console David Kopf Model 942 Surgical equipment
Non-rupture Ear Bars, set of 2 (Rat) David Kopf Model 955 Surgical equipment
Anesthesia Mask (Rat) David Kopf Model 906 Surgical equipment
High speed Stereotaxic drill includes table top motor controller, foot pedal, handpiece, stereotaxic handpiece holder David Kopf Model 1474 Surgical equipment
Microdrill burrs, 0.9 mm Fine Science Tools Inc 19007-09 Surgical supply
Automated Syringe pump with Micro4 Controller  David Kopf Model UMP3-1 Surgical equipment
Pro-animal detachable Ceramic Blade Clipper Kit Ahdis 21420 Surgical supply
Betadine Skin Cleanser Perdue  Products L.P 67618-149-04 Surgical supply
Triple Antiobiotic Ointment Medline Supply 53329-087-01 Surgical supply
Puralube Vet Ointment Only Veterinary Supply 17033-211-38 Surgical supply
Dino-lite  Microscope AD7013MTL An alternative to the traditional disection scope
Dino-lite Rigid TableTop Boom Stand Microscope MS36B Surgical equipment
28 Gauge 10uL Syringe Hamilton  80308-701SN Surgical equipment
Extra Tall MDF Sound attenuating Cubicle Med Associates, Inc ENV-018MD 22'Wx22"Hx16"D
Extra Tall Modular Test Chamber Med Associates, Inc ENV-007 Behavioral equipment
Stainless Steel Grid Floor  Med Associates, Inc ENV-005 Behavioral equipment
House Light Med Associates, Inc ENV-215M Used as the house light and stimulus light
Modular Pellet Dispenser  Med Associates, Inc ENV-203M-45 Behavioral equipment
Pellet Recetacle, Cup Type Med Associates, Inc ENV-200R1M Behavioral equipment
Head Entry Detector for Rat Med Associates, Inc ENV-254-CB Behavioral equipment
Dustless precision food pellets, 45 mg Bio-Serv F0165 Behavioral supply
Cage Speaker for Rat Chamber Med Associates, Inc ENV-224AM Behavioral equipment
Programmable Audio Generator  Med Associates, Inc ANL-926 Behavioral equipment
Smart Ctrl 8 Input/16 output Package Med Associates, Inc DIG-716P2 Behavioral equipment
Large Table Top Cabinet and Power Supply Med Associates, Inc SG-6510D Behavioral equipment
PCI Interface Package Med Associates, Inc DIG-700P2-R2 Behavioral equipment
MED Intel core Computer Pkg with X Pro 19" Monitor Med Associates, Inc COM-103V Behavioral equipment
Paraformaldehyde (grannular), 1 kg Electron Microsopy Sciences 19210 Hazard:  carcinogen, weigh in hood
Rabbit Monoclonal antibody (HA-Tag) Cell Signaling Technologies  3724S Histology reagent
XP Rabbit monoclonal antibody (GFP) Cell Signaling Technologies  2956S Histology reagent
Anti-Rabbit IgG Cell Signaling Technologies  4412S Histology supplies
Superfrost Plus slides  VWR international 483111-703 Histology supplies

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Cite This Article
Robinson, S., Adelman, J. S. A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning. J. Vis. Exp. (100), e52859, doi:10.3791/52859 (2015).

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