调整在海马Theta乐队<em>体外</em>：从孤立的啮齿动物皮下注射电路记录的方法
Summary
在这里，我们提出了从孤立的整个海马制剂记录节律神经元网络theta和γ振荡的协议。我们描述从海马提取到场，单元和全细胞膜片钳记录的细节以及θ节律的光起搏的实验步骤。
Abstract
该方案概述了从孤立的整个海马，WT和转基因小鼠制备和记录的程序，以及近来对θ振荡研究的方法和应用的改进。提出了分离的海马制剂的简单表征，其中检查内部海马θ振荡器之间的关系以及玉米螟-1（CA1）和亚细胞（SUB）区域的锥体细胞和GABA能中间神经元的活性。总体而言，我们显示分离的海马能够在体外产生内在的θ振荡，海马内产生的节律性可以通过对小白蛋白阳性（PV）中间神经元的光诱导刺激进行精确的操纵。 体外分离的海马制剂提供了独特的机会，使用来自视觉识别的neu的同时场和细胞内膜片钳记录更好地了解θ节奏生成的机制。
Introduction
海马THETA振荡（4 – 12赫兹）之间是在哺乳动物脑节律活动的最主要的形式，并且据信在认知功能中发挥关键作用，如中的情节记忆1，2，3时空信息和形成处理。虽然一些体内研究，突出THETA调制地方细胞具有空间导航和病变的研究，以及临床证据的关系，支持海马THETA振荡参与记忆形成4，5，6，相关联的机制视图伴随着海马theta振荡的产生尚未完全了解。早期体内研究表明，θ活性主要依赖于外在振荡器，特别是节奏输入从传入大脑结构，例如隔膜和内嗅皮层7，8，9，10。与海马神经元的属性一起海马神经网络的内部连通- -一种内在因素作用也假定的基于体外观察11，12，13，14，15，16，17，18。然而，除了少数标志性开发方法研究19，20，21，困难可以复制的简单体外切片准备生理现实群体活动长期以来，延迟了对海马和相关领域的内在能力进行更为详细的实验检查来自我产生θ振荡。
标准体外薄片实验设置的一个重要缺点是脑结构的3D细胞和突触组织通常受到损害。这意味着不能支持基于空间分布的细胞组件的许多形式的协调网络活动，范围从局部组（半径≤1mm）到遍及一个或多个脑区域（> 1mm）的神经元群体。考虑到这些考虑，需要一种不同类型的方法来研究θ振荡如何在海马中出现并传播到相关的皮层和皮质下输出结构。
近年来，初步开展了“完整的海马”准备，以检查双向intera两个结构22和“分离的海马”制剂的随后演变ctions，已经揭示，在缺少外部节奏输入23海马自发发生固有THETA振荡。这些方法的价值在于初步的认识，即这些区域的整个功能结构必须被保留以便在体外作为θ节律发生器22 。
Protocol
所有程序均按照麦吉尔大学动物保护委员会和加拿大动物保护委员会批准的议定书和准则进行。 急性海马体外制备注意：分离完整的海马制剂涉及三个主要步骤：（1）溶液和设备的制备，（2）海马的解剖和（3）建立产生内在θ振荡所需的快速灌注速率系统。在该方案中，及时执行手术 – 从解剖到记录 – 特别重要，因为分离的海马构成如此密集但精?…
Representative Results
本节说明通过在体外研究小鼠分离的海马制剂中的 θ振荡可以获得的结果的实例。提取分离的海马的解剖程序如图1所示 。使用这种制备，可以在放置多个场电极期间检查内在θ振荡，记录整体活动和将突触输入同步到分离的海马的不同区域和层中的神经元群体（ 图2 ）。提出了同时进行的全细胞膜片钳和细胞…
Discussion
虽然来自急性海马切片的电生理记录构成了体外标准技术，但是本文提出的方法与传统方法有很大不同。不同于其中特定细胞层在表面可见并且可以直接检查的薄片制剂，完整的海马制剂更类似于体内构型，其中电极在穿过单个层时下降到目标脑区域中。海马的完整性与局部神经元群体的功能连通性和性质一起保存。这提供了一个复杂而强大的工具，用于调查海马中的小型和大型网?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加拿大卫生研究与自然科学研究所的支持。
Materials
| Reagents | |||
| Sodium Chloride | Sigma Aldrich | S9625 | |
| Sucrose | Sigma Aldrich | S9378 | |
| Sodium Bicarbonate | Sigma Aldrich | S5761 | |
| NaH2PO4 – sodium phosphate monobasic | Sigma Aldrich | S8282 | |
| Magnesium sulfate | Sigma Aldrich | M7506 | |
| Potassium Chloride | Sigma Aldrich | P3911 | |
| D-(+)-Glucose | Sigma Aldrich | G7528 | |
| Calcium chloride dihydrate | Sigma Aldrich | C5080 | |
| Sodium Ascorbate | Sigma Aldrich | A7631-25G | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Standard Dissecting Scissors | Fisher Scientific | 08-951-25 | brain extraction |
| Scalpel Handle #4, 14cm | WPI | 500237 | brain extraction |
| Filter forceps, flat jaws, straight (11cm) | WPI | 500456 | brain extraction |
| Paragon Stainless Steel Scalpel Blades #20 | Ultident | 02-90010-20 | brain extraction |
| Fine Point Curved Dissecting Scissors | Thermo Fisher Scientific | 711999 | brain extraction |
| Teflon (PTFE) -coated thin spatula | VWR | 82027-534 | hippocampal preparation |
| Hayman Style Microspatula | Fisher Scientific | 21-401-25A | hippocampal preparation |
| Lab spoon | Fisher Scientific | 14-375-20 | hippocampal preparation |
| Borosilicate Glass Pasteur Pipets | Fisher Scientific | 13-678-20A | hippocampal preparation |
| Droper | Fisher Scientific | hippocampal preparation | |
| Razor blades Single edged | VWR | 55411-055 | hippocampal preparation |
| Lens paper (4X6 inch) | VWR | 52846-001 | hippocampal preparation |
| Glass petri dishes (100 x 20 mm) | VWR | 25354-080 | hippocampal preparation |
| Plastic tray for ice; size 30 x 20 x 5 cm | n.a. | n.a. | hippocampal preparation |
| Single Inline Solution Heater | Warner Instruments | SH-27B | perfusion system |
| Aquarium air stones for bubbling | n.a. | n.a. | perfusion system |
| Tygon E-3603 tubing (ID 1/16 OD 1/8) | Fisherbrand | 14-171-129 | perfusion system |
| Electric Skillet | Black & Decker | n.a. | perfusion system |
| 95% O2/5% CO2 gas mixture (carbogen) | Vitalaire | SG466204A | perfusion system |
| Glass bottles/flasks (4 x 1 L) | n.a. | n.a. | perfusion system |
| Submerged recording Chamber | custom design (FM) | n.a. | Commercial alternative may be used |
| Glass pipettes (1.5 / 0.84 OD/ID (mm) ) | WPI | 1B150F-4 | electrophysiology |
| Hum Bug 50/60 Hz Noise Eliminator | Quest Scientific | Q-Humbug | electrophysiology |
| Multiclamp 700B patch-clamp amplifier | Molecular devices | MULTICLAMP | electrophysiology |
| Multiclamp 700B Commander Program | Molecular devices | MULTICLAMP | electrophysiology |
| Digital/Analogue converter | Molecular devices | DDI440 | electrophysiology |
| PCLAMP10 | Molecular devices | PCLAMP10 | electrophysiology |
| Vibration isolation table | Newport | n.a. | electrophysiology |
| Micromanipulators (manually operated ) | Siskiyou | MX130 | electrophysiology (LFP) |
| Micromanipulators (automated) | Siskiyou | MC1000e | electrophysiology (patch) |
| Audio monitor | A-M Systems | Model 3300 | electrophysiology |
| Micropipette/Patch pipette puller | Sutter | P-97 | electrophysiology |
| Custom-built upright fluorescence microscope | Siskiyou | n.a. | Imaging |
| Analogue video camera | COHU | 4912-2000/0000 | Imaging |
| Digital frame grabber with imaging software | EPIX, Inc | PIXCI-SV7 | Imaging |
| Olympus 2.5x objective | Olympus | MPLFLN | Imaging |
| Olympus 40x water immersion objective | Olympus | UIS2 LUMPLFLN | Imaging |
| Custom-made light-emitting diode (LED) system | custom | n.a. | optogenetic stimulation (Amhilon et al., 2015) |
| Name | Company | Catalog Number | Comments |
| Animals | |||
| PV::Cre (KI) mice | Jackson Laboratory | stock number 008069 | Allow Cre-directed gene expression in PV interneurons |
| Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI)) | Jackson Laboratory | stock number 007905 | Express TdTomato following Cre-mediated recombination |
| Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP | Jackson Laboratory | stock number 012569 |
Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase |
| PVChY mice | In house breeding | n.a. | Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP |
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Citazione di questo articolo
Manseau, F., Williams, S. Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit. J. Vis. Exp. (126), e55851, doi:10.3791/55851 (2017).