アラート、頭部拘束ラットの皮質下脳構造内の単一ニューロンのJuxtacellular監視とローカリゼーション
Summary
This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.
Abstract
There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal’s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.
Introduction
積極的に行動のタスクに従事し、アラート、動物における神経活動の監視は、神経系の機能と組織を理解するために重要です。シングルニューロンユニットからの電気的活動の細胞外記録は、長いシステム神経科学の定番ツールとなって、現在で使用されて広くまだいる。電極の種類とさまざまな構成は、特定の実験の科学的·技術的な要求に応じて利用可能です。慢性移植マイクロドライブまたは電極アレイは、多くの場合、鳥類、げっ歯類、非ヒト霊長類を含む1-4、自由に動物を移動させる際に使用される。また、外部のマイクロマニピュレーターを経由して金属やガラス微小電極を伴う急性の貫通部は、多くの場合、麻酔をかけたり頭拘束動物から記録するために使用されます。ガラスマイクロピペット電極は、それらが明確に隔離するjuxtacellularまたは「細胞添付」構成で使用することができるという利点を有する5ソート事後スパイクの合併症のない単一ニューロンの活動。これらは、色素または神経解剖学的トレーサーの小さな堆積物を注入するために、または個々のセルを記録充填するために使用することができるようにこれらの電極はさらに、解剖学的に同定された細胞または場所から記録を可能にする。この構成は、正常に、ラット、マウス、鳥類6-10に適用されている。現在記載の技術は、juxtacellular監視や警告における細胞外の色素沈着、頭部拘束ラットに焦点を当てています。埋めjuxtacellular単一のセルとは異なり、これらの色素の沈着は細胞形態または軸索突起11についての情報を提供していないが、彼 らは約50μmの正確な解剖学的局在を有効にし、批判的に、アラートの動物では有意に高い収量を持っていることに注意してください。それにもかかわらず、解剖学的標識のための代替戦略として提供されて埋めjuxtacellular単一セルに関する情報。
要するに、プロトコルは3主要な段階で構成されています。第一段階では、ラットを6日間にわたって布靴下（ 図1）身体拘束に順応される。第2段階では、ヘッドレスト装置（ 図2）および記録チャンバーは、外科的に、ラットは、複数の次の記録セッション（ 図3）の間に定位面内に維持することができるように注入される。この手順は、標準的な参照が12を座標に基づいて、電気生理学的研究のために、脳の特定のサブ皮質領域を標的とするように実験者が可能になります。第三相は、確実に1つのユニット6-9を分離juxtacellularニューロンの記録を、（ 図5）石英キャピラリー管から電極を構成し、行動および電気生理学的実験（ 図4）を行って製造するための適切な治具にラットを配置することを含む、および解剖locatiマーキングシカゴスカイブルー色素（ 図6および7）と記録部位の上。録音は、同時行動の監視を行っている。しかし、行動の技術的な詳細は、各実験の科学的な目標に依存し、単一のプロトコルの範囲を超えてこのようにある。複数の日に繰り返すことができる実験手順の完了後、動物を安楽死させる。脳は、明視野または蛍光顕微鏡のいずれかを使用して、標準的な神経解剖技術に従って抽出され、処理される。
Protocol
Representative Results
Discussion
実験的な治具の構築
それは、様々な方法で構築することができるように、実験治具（ 図4）を構築するために使用される機械部品の説明は、プロトコルから省略されている。このデモでは、標準的な光学機械部品とサポートクランプは（ 材料の項を参照）ヘッドレストバーと身体拘束チューブを取り付けるために使用さ?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.
Materials
| Name of the Reagent | Company | Catalogue Number | Comments |
| Ketaset (Ketamine HCl) | Fort Dodge | N/A | |
| Anased (Xylazine solution) | Lloyd Laboratories | N/A | |
| Betadyne (Povidone-Iodine) | CVS Pharmacy | 269281 | |
| Loctite 495 | Grainger Industrial Supply | 4KL86 | 20-40 cp cyanoacrylate |
| Vetbond | 3M | 1469SB | |
| Grip cement powder | Dentsply Intl | 675571 | For the base of the recording chamber |
| Grip cement liquid | Dentsply Intl | 675572 | For the base of the recording chamber |
| Silicone Gel | Dow Corning | Mar-80 | |
| Jet denture repair acrylic powder | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Ortho-Jet Fast curing orthodontic acrylic resin liquid | Lang Dental Manufacturing Co. | N/A | For securing the head restraint apparatus to the cranium |
| Chicago sky blue | Sigma | C8679 | |
| Paraformaldehyde | Sigma | 158127 | For perfusion and tissue fixation |
| Phosphate-buffered saline | Sigma | P3813 | For perfusion and tissue fixation |
| Cytochrome C | Sigma | C2506 | For cytochrome-oxidase staining (Figure 7) |
| Diaminobenzidine | Sigma | D5905 | For cytochrome-oxidase staining (Figure 7) |
| Material Name | Company | Catalogue Number | Comments |
| Rat sock | Sew Elegant (San Diego, CA) | N/A | Custom made, Figures 1, 4 |
| PVC tube 2 ½” | U.S. Plastic Co. | 34108 | Figure 4 |
| Subminiature D pins & sockets | TE Connectivity | 205089-1 | Figure 3 |
| Stainless steel music wire 0.010” diameter | Precision Brand Products, Inc. | 21010 | Figure 3 |
| Stereotaxic holding frame | Kopf Instruments | Model 900 | Figure 3 |
| Stereotaxic ear bars | Kopf Instruments | Model 957 | Figure 3 |
| Stereotaxic manipulator | Kopf Instruments | Model 960 | Figure 3 |
| ½ mm drill burr | Henry Schein | 100-3995 | |
| Quiet-Air dental drill | Midwest Dental | 393-1600 | |
| Stainless steel 0-80 1/8” screw | Fastener superstore | 247438 | Figure 3 |
| 0.2mL centrifuge tube | Fisher Scientific | 05-407-8A | Figure 3 |
| Custom head-holding bar | UCSD SIO Machine Shop | N/A | Custom made, Figures 2, 3, 4 |
| Custom head-holding plate | UCSD SIO Machine Shop | N/A | Custom made, Figure 2, 3, 4 |
| Right angle post-clamp | Newport | MCA-1 | Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp. |
| 8-32 3/4” screw | Fastener Superstore | 240181 | For head-restraint, Figure 3 |
| 4-40 ¼” screw | Fastener Superstore | 239958 | For head restraint, Figures 3, 4 |
| Quartz capillary tubing | Sutter Instruments | QF-100-60-10 | Figure 5 |
| Carbon dioxide laser puller | Sutter instruments | P-2000 | |
| Motorized micromanipulator | Sutter Instruments | MP-285 | |
| Microelectrode amplifier | Molecular Devices | Multiclamp 700B | Alternate part: Molecular Devices Axoclamp 900A |
| Microelectrode amplifier head stage | Molecular Devices | CV-7B | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Isolated pulse stimulator | A-M Systems | Model 2100 | Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A |
| Audio monitor | Radio Shack | 32-2040 | |
| Pipette holder | Warner Instruments | #MEW-F10T | Alternate parts: see Discussion |
| Figure 6A | |||
| Electrode lead wire | Cooner wire | NEF34-1646 | (optional), Figure 6D |
| Relay for amplifier head-stage | COTO Technology | #2342-05-000 | (optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C |
| Digital video camera | Basler | A602fm | (optional) For behavioral monitoring, Figure 7 |
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