著者はスティーブ・ジーゲルバウムのサポートに感謝したいと思います。資金は5R01NS106983-02と1 F31 NS113466-01によって提供されます。

著者は開示するものは何もない。

組織の健康を促進し、自発的な自然主義的なネットワーク活動の出現を支持するように設計されたこのスライスプロトコルにはいくつかのステップがあります:マウスは、チルドショ糖切断溶液で経皮的に浸透しています。水平内膜皮質(HEC)スライスは、中間または腹側海馬から450μmの厚さで切断されます。スライスは、温めたACSFのインターフェースで回復し、加湿された、カルボゲンが豊富...

生体外の生きた海馬スライスからの電気生理学的測定は、多くの利点を有する強力な実験的アプローチである。実験者は顕微鏡、マイクロマニピュレーター、および記録システムを使用して、組織内の個々のニューロンから測定値を直接可視化し、収集することができます。組織スライスは光遺伝学的、化学遺伝学的、または薬理学的実験のための光刺激または薬物送達にも非常にアクセス可能である。
海馬ネットワークは、生体内で高度に同期的な集団活動を生成し、細胞外局所的なフィールドポテンシャル1、2、3、4、5の振動として見える。脳スライス法は、これらの神経ネットワーク振動の根底にある細胞および回路メカニズムに関する洞察を得るために活用されてきた。Maierらの基礎的な研究は、鋭波波波紋成(SRS)が腹側海馬6,7のスライスに自発的に出現できることを実証した。複数の研究者からのその後の研究は、海馬8、9、10のネットワーク状態を調節する神経調節因子の役割およびvivo11での行動の間に以前に活動していた神経アンサンブルのインビトロ再活性化を促進するシナプス機構を含む、SDRの多くの側面を徐々に解明してきた。脳スライス実験はまた、ガンマ範囲振動(30〜100Hz)、記憶符号化とリコール12、13をサポートすると考えられている明確な海馬ネットワーク状態への洞察を提供している。最後に、側頭葉てんかん14,15の病態生理学における海馬および関連する構造の中心的役割を認識し、研究者は海馬スライス製剤を使用しててんかん活動の発生および伝播を調査した。Carterららは、慢性てんかん動物から調製された海馬-内皮質スライスを組み合わせることで、vitro16で自発的にてんかん放電を発生させることができることを実証した。その後、Karlócaiらは、改変されたイオン濃度(Mg2+または上昇K+)または薬物(4APまたはガバジン)17を有する改変された人工脳脊髄液(ACSF)を用いて海馬スライス中のてんかん分泌物の基礎となるメカニズムを探った。
研究者は、重要な方法で異なる多数の海馬スライスアプローチを開発しました:(1)スライスに含まれる海馬の領域(側線、中間体、または腹側);(2)内皮質などの海馬外組織の有無。(3)スライス(コロナ、矢状、水平、または斜め)をカットするために使用される方向。(4)スライス後に組織が維持される条件(ACSFに完全に沈水するか、ACSFのインターフェースで保持され、加湿された、カルボゲンが豊富な空気)。
どのスライスアプローチを使用するかを選択することは、実験目的によって決定されるべきである。例えば、水没条件下で維持された眼海馬の横方向またはコロナスライスは、海馬内回路およびシナプス可塑性18、19、20の調査に非常に有効に使用されてきた。しかし、このような調製物は、腹側海馬21、22、23からのスライスほど容易にネットワーク振動を発生させるものではない。持続的なSWR活性の状態は、背側および腹側海馬24からの横切りスライスにおけるテタニック刺激によって誘発され得るが、腹側スライス7,25において自発的なSWRがより容易に観察される。
側方海馬と腹側海馬の間に固有の生理学的および解剖学的な区別は、生体内およびインビトロ26の両方で行われる研究によって支えられている。ラットにおける記録は、側腹および中間海馬全体にわたって強く一貫したシータリズムを明らかにしたが、腹側領域と海馬27の残りの部分との間の一貫性が悪い。生体内のSRSは、裏側海馬と中間海馬の間で容易に伝播し、腹側海馬に由来するSDRはしばしば局所的な28のままである。海馬の縦軸に沿って、裏側および中間海馬に存在するCA3錐体ニューロンに由来する関連投影法。腹側領域から発生するCA3投影は比較的局所的であり、したがって、スライスプロセス29、30の間に切断される可能性が低い。したがって、腹側スライスは、人口同期を生成するために必要な繰り返しネットワークをよりよく保存する可能性があります。vitroで自発的なネットワーク活動を発生させる腹側スライスの傾向は、より多くの側側領域31と比較して、錐体ニューロンの高い固有興奮性または腹側海馬におけるGABAergic阻害の高い固有の興奮性を反映してもよい。確かに、腹側海馬のスライスは、てんかん活動32、33に対してより敏感である。したがって、自発的な生理学的8、9、11、24または病理学的16、34、35、36ネットワーク振動の多くの研究は、伝統的に、時には腹側海馬の横面に平行な組織スライスを生み出す前頭後頭方向にわずかな角度で、水平スライスアプローチを使用してきました。
スライス処理によって、スライスの多くのセルが切断されることで、ネットワーク接続が不可避的に影響を受けます。目的の回路の接続性を最適化するために、準備中に保持されるスライスと組織の角度と厚さを考慮する必要があります。多くの研究は、生理学的または病理学的ネットワーク振動の文脈で2つの構造間の相互作用を探求するために水平結合海馬-内皮質スライス(HEC)を利用しています。Rothらは、海馬のCA1サブフィールドと内側の内皮質の層Vから二重記録を行い、HECスライス37を通してSWR活性の伝播を実証した。てんかんの活動の多くの研究は、てんかんの排出がコルチコヒッポカンネットワーク16、35、36、38を介して伝播する方法を調査するためにHECスライス調製を使用しています。無傷のコルチコヒッポカンピカルループの保存は、自発的なSVR、てんかんの排出、またはガンマ振動の前提条件ではないことを注意することが重要です。ネットワーク振動は、付着した海馬組織21、22、23、25、39、40、41を伴わない、側側または腹側海馬の横切れスライスで生成することができる。海馬スライスにおけるネットワーク振動の自発的発生のためのより重要な要因は、厚いスライス(400〜550 μm)がCA2/CA3の再発ネットワーク21、22、25でより多くの接続を維持するので、各スライスの厚さである可能性があります。
角度付き水平HECスライス(前頭部後頭部方向に約12°の角度でカット)は、コルチコヒッポカンピカルループ11、16、34、35、42の機能的接続性を研究するために使用されてきたが、そのような斜めの準備は、自発的なネットワーク活動43、44、45のために必要とされない。しかし、斜めのスライス平面を使用すると、下向きまたは上向きの角度が適用されるかに応じて、研究者が腹側または中間海馬の横方向の層板を最もよく保存するスライスを選択的に作成することができます(図1)。このアプローチは、パパテオドロプロスら、2002年に使用される方法と概念的に似ており、各海馬を自由に解剖し、組織チョッパーを使用して、側腹側全軸21に沿って横切りスライスを作成した。腹側と側側中海馬の機能的な区別に照らして、研究者は実験を設計したり結果を解釈したりする際に、スライスの解剖学的起源を考慮する必要があります。スライス手順中に寒天ランプを使用することは、中間海馬または腹側海馬のいずれかからスライスを優先的に生成する簡単な方法です。
海馬スライスは、水没したチャンバー(組織がACSFに完全に浸漬された)またはインターフェイススタイルのチャンバー(例えば、オスロまたはハースチャンバー、流れる媒体の薄膜のみで覆われたスライス)のいずれかで維持することができます。インターフェースの維持は、組織の酸素化を促進し、神経細胞の生存を促進し、ニューロン間活動の持続的な高レベルを可能にする。従来、沈められた記録条件は、ネットワークレベル振動の安定した発現のために十分な組織酸素化を提供しない、より遅いACSF流量を使用する。水没した海馬スライスでは、カルバコール誘発ガンマ振動は一過性に観察されるだけであるが、それらは界面記録室10、48、49で安定的に維持することができる。このように、vitroの複雑な自発的活動の多くの研究は、鋭波波波の複合体6、7、8、9、10、25、37、ガンマ振動10、38、45、47を調査するためにインターフェース記録室に依存してきました。
水没式記録室では、浸漬顕微鏡の目的を使用して個々の細胞を可視化し、記録のために健康的な細胞を選択的にターゲットにすることができます。水没した製剤はまた、水没が組織への薬物または他の化合物の急速な拡散を促進するので、細胞ミリューを細かく制御することを可能にする。したがって、水没条件下で安定したネットワーク振動が維持される修正方法論は、強力な実験アプローチを表す。このアプローチは、組織12、48、49への酸素供給を増強するためにACSF(〜6 mL/min)の高い流量を有する改変された水没記録室に移る前に、海馬スライスが簡略化されたインターフェーススタイルの保持室で数時間回復するハホスらの働きによって例示される。これらの条件下では、高レベルのニューロン間活動と安定した自発的ネットワーク振動を、水中記録室に維持することができる。この変更アプローチにより、研究者は視覚的に誘導された全細胞パッチクランプ記録を行い、形態学的に同定された細胞タイプがカルバコール誘導ガンマ振動12に寄与することを特徴付けることを可能にする。SRS は、ACSF11、48、49の速い流量を持つ水没した海馬スライスでも自然発生する可能性があります。Maierら et al. 水没した記録室に移る前にインターフェース室で回収された海馬スライスが確実に自発的なSWRを示したのに対し、水没した記録室に移る前にビーカーに沈んだスライスは、より小さな呼び起動場応答を示し、自発的なシナプス電流のレベルが低く、自発的なSWR43を示すことはほとんどありませんでした。Schlingloffらは、この改良された方法論を用いて、自発的なSRS44の生成におけるパルブアルブミン発現バスケット細胞の役割を実証した。
次のプロトコルは、水平海馬スライス中の自発的に活動するニューロンを界面条件下で回収し、その後、薬理学的または光遺伝学的操作および視覚的に導かれた記録に適した水没記録室で維持することができるスライス法を提示する。

<table><tbody><tr><td>3D printer</td><td>Lulzbot</td><td>LulzBot TAZ 6</td><td></td></tr><tr><td>Acute brain slice incubation holder</td><td>NIH 3D Print Exchange</td><td>3DPX-001623</td><td>Designed by ChiaMing Lee, available at https://3dprint.nih.gov/discover/3dpx-001623</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate magnesium salt</td><td>Sigma Aldrich</td><td>A9187-500MG</td><td></td></tr><tr><td>Ag-Cl ground pellets</td><td>Warner</td><td>64-1309, (E205)</td><td></td></tr><tr><td>agar</td><td>Becton, Dickinson</td><td>214530-500g</td><td></td></tr><tr><td>ascorbic acid</td><td>Alfa Aesar</td><td>36237</td><td></td></tr><tr><td>beaker (250 mL)</td><td>Kimax</td><td>14000-250</td><td></td></tr><tr><td>beaker (400 mL)</td><td>Kimax</td><td>14000-400</td><td></td></tr><tr><td>biocytin</td><td>Sigma Aldrich</td><td>B4261</td><td></td></tr><tr><td>blender</td><td>Oster</td><td>BRLY07-B00-NP0</td><td></td></tr><tr><td>Bonn scissors, small</td><td>becton, Dickinson</td><td>14184-09</td><td></td></tr><tr><td>borosilicate glass capillaries with filament (O.D. 1.5 mm, I.D. 0.86 mm, length 10 cm)</td><td>Sutter Instruments</td><td>BF150-86-10HP</td><td>Fire polished capillaries are preferable.</td></tr><tr><td>calcium chloride solution (1 M)</td><td>G-Biosciences</td><td>R040</td><td></td></tr><tr><td>camera</td><td>Olympus</td><td>OLY-150</td><td></td></tr><tr><td>compressed carbogen gas (95% oxygen / 5% carbon dioxide)</td><td>Airgas</td><td>X02OX95C2003102</td><td></td></tr><tr><td>compressed oxygen</td><td>Airgas</td><td>OX 200</td><td></td></tr><tr><td>constant voltage isolated stimulator</td><td>Digitimer Ltd.</td><td>DS2A-Mk.II</td><td></td></tr><tr><td>coverslips (22x50 mm)</td><td>VWR</td><td>16004-314</td><td></td></tr><tr><td>cyanoacrylate adhesive</td><td>Krazy Glue</td><td>KG925</td><td>Ideally use the brush-on form for precision</td></tr><tr><td>data acquisition software</td><td>Axograph</td><td>N/A</td><td>Any equivalent software (e.g. pClamp) would work.</td></tr><tr><td>Dell Precision T1500 Tower Workstation Desktop</td><td>Dell</td><td>N/A</td><td>Catalog number will depend on specific computer - any computer will work as long as it can run electrophysiology acquisition software.</td></tr><tr><td>Digidata 1440A</td><td>Molecular Devices</td><td>1-2950-0367</td><td></td></tr><tr><td>digital timer</td><td>VWR</td><td>62344-641</td><td>4-channel Traceable timer</td></tr><tr><td>disposable absorbant pads</td><td>VWR</td><td>56616-018</td><td></td></tr><tr><td>dissector scissors</td><td>Fine Science Tools</td><td>14082-09</td><td></td></tr><tr><td>double-edge razor blades</td><td>Personna</td><td>BP9020</td><td></td></tr><tr><td>dual automatic temperature controller</td><td>Warner Instrument Corporation</td><td>TC-344B</td><td></td></tr><tr><td>dual-surface or laminar-flow optimized recording chamber</td><td>N/A</td><td>N/A</td><td>The chamber presented in this protocol is custom made. A commercial equivalent would be the RC-27L from Warner Instruments.</td></tr><tr><td>equipment rack</td><td>Automate Scientific</td><td>FR-EQ70&quot;</td><td>A rack is not strictly necessary but useful for organizing electrophysiology</td></tr><tr><td>Ethylene glycol-bis(2-aminoethyiether)- N,N,N&#039;,N&#039;-teetraacetic acid (EGTA)</td><td>Sigma Aldrich</td><td>324626-25GM</td><td></td></tr><tr><td>filter paper</td><td>Whatman</td><td>1004 070</td><td></td></tr><tr><td>fine scale</td><td>Mettler Toledo</td><td>XS204DR</td><td></td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>glass petri dish (100 x 15 mm)</td><td>Corning</td><td>3160-101</td><td></td></tr><tr><td>glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Guanosine 5&amp;prime;-triphosphate sodium salt hydrate</td><td>Sigma Aldrich</td><td>G8877-250MG</td><td></td></tr><tr><td>ice buckets</td><td>Sigma Aldrich</td><td>BAM168072002-1EA</td><td></td></tr><tr><td>isoflurane vaporizer</td><td>General Anesthetic Services</td><td>Tec 3</td><td></td></tr><tr><td>lab tape</td><td>Fisher Scientific</td><td>15-901-10R</td><td></td></tr><tr><td>lens paper</td><td>Fisher Scientific</td><td>11-996</td><td></td></tr><tr><td>light source</td><td>Olympus</td><td>TH4-100</td><td></td></tr><tr><td>magnesium chloride solution (1 M)</td><td>Quality Biological</td><td>351-033-721EA</td><td></td></tr><tr><td>magnetic stir bars</td><td>Fisher Scientific</td><td>14-513-56</td><td>Catalog number will be dependent on the size of the stir bar.</td></tr><tr><td>micromanipulator</td><td>Luigs &amp;amp; Neumann</td><td>SM-5</td><td></td></tr><tr><td>micromanipulator (manual)</td><td>Scientifica</td><td>LBM-2000-00</td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>microspatula</td><td>Fine Science Tools</td><td>10089-11</td><td></td></tr><tr><td>monitor</td><td>Dell</td><td>2007FPb</td><td></td></tr><tr><td>MultiClamp 700B Microelectrode Amplifier</td><td>Molecular Devices</td><td>MULTICLAMP 700B</td><td>The MultiClamp 700B should include headstages, pipette holders, and a model cell.</td></tr><tr><td>N-(2-Hydroxyethyl)piperazine-N&amp;prime;-(2-ethanesulfonic acid), (HEPES)</td><td>Sigma Aldrich</td><td>H3375-25G</td><td></td></tr><tr><td>needle (20 gauge, 1.5 in length)</td><td>Becton, Dickinson</td><td>305176</td><td></td></tr><tr><td>nylon filament</td><td>YLI Wonder Invisible Thread</td><td>212-15-004</td><td>size 0.004. This cat. # is from Amazon.com</td></tr><tr><td>nylon mesh</td><td>Warner Instruments Corporation</td><td>64-0198</td><td></td></tr><tr><td>perstaltic pump</td><td>Harvard Apparatus</td><td>70-2027</td><td></td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma Aldrich</td><td>P1937-1G</td><td></td></tr><tr><td>pipette holders</td><td>Molecular Devices</td><td>1-HL-U</td><td></td></tr><tr><td>platinum wire</td><td>World Precision</td><td>PT0203</td><td></td></tr><tr><td>polylactic acid (PLA) filament</td><td>Ultimaker</td><td>RAL 9010</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma Aldrich</td><td>P3911-500G</td><td></td></tr><tr><td>potassium gluconate</td><td>Sigma Aldrich</td><td>1550001-200MG</td><td></td></tr><tr><td>potassium hydroxide</td><td>Sigma Aldrich</td><td>60377-1KG</td><td></td></tr><tr><td>razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>roller clamp</td><td>World Precision Instruments</td><td>14041</td><td></td></tr><tr><td>scale</td><td>Mettler Toledo</td><td>PM2000</td><td></td></tr><tr><td>scalpel handle</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>slice harp</td><td>Warner</td><td>SHD-26GH/2</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Fisher Chemical</td><td>S233-500</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma Aldrich</td><td>S9888-1KG</td><td></td></tr><tr><td>sodium phosphate monobasic anhydrous</td><td>Fisher Chemical</td><td>S369-500</td><td></td></tr><tr><td>sodium pyruvate</td><td>Fisher Chemical</td><td>BP356-100</td><td></td></tr><tr><td>spatula</td><td>VWR</td><td>82027-520</td><td></td></tr><tr><td>spatula/spoon, large</td><td>VWR</td><td>470149-442</td><td></td></tr><tr><td>sterile scalpel blades</td><td>Feather</td><td>72044-10</td><td></td></tr><tr><td>stirrer / hot plate</td><td>Corning</td><td>6795-220</td><td></td></tr><tr><td>stopcock valves, 1-way</td><td>World Precision Instruments</td><td>14054</td><td></td></tr><tr><td>stopcock valves, 3-way</td><td>World Precision Instruments</td><td>14036</td><td></td></tr><tr><td>sucrose</td><td>Acros Organics</td><td>AC177142500</td><td></td></tr><tr><td>support for swivel clamps</td><td>Fisher Scientific</td><td>14-679Q</td><td></td></tr><tr><td>surgical scissors, sharp/blunt</td><td>Fine Science Tools</td><td>14001-12</td><td></td></tr><tr><td>syringe (1 mL)</td><td>Becton, Dickinson</td><td>309659</td><td></td></tr><tr><td>syringe (60 mL with Luer-Lok tip)</td><td>Becton, Dickinson</td><td>309653</td><td></td></tr><tr><td>three-pronged clamp</td><td>Fisher Scientific</td><td>05-769-8Q</td><td></td></tr><tr><td>tissue forceps, large</td><td>Fine Science Tools</td><td>11021-15</td><td></td></tr><tr><td>tissue forceps, small</td><td>Fine Science Tools</td><td>11023-10</td><td></td></tr><tr><td>transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>tubing</td><td>Tygon</td><td>E-3603</td><td>ID 1/16 inch, OD 3/16 inch</td></tr><tr><td>tubing</td><td>Tygon</td><td>R-3603</td><td>ID 1/8 inch, OD 1/4 inch</td></tr><tr><td>vacuum grease</td><td>Dow Corning</td><td>14-635-5D</td><td></td></tr><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT 1200S</td><td></td></tr><tr><td>vibration-dampening table with faraday cage</td><td>Micro-G / TMC-ametek</td><td>2536-516-4-30PE</td><td></td></tr><tr><td>volumetric flask (1 L)</td><td>Kimax</td><td>KIM-28014-1000</td><td></td></tr><tr><td>volumetric flask (2 L)</td><td>PYREX</td><td>65640-2000</td><td></td></tr><tr><td>warm water bath</td><td>VWR</td><td>1209&lt;br/&gt; &amp;nbsp;</td><td></td></tr></tbody></table>

acute mouse brain slicing to investigate spontaneous hippocampal network activity

急性げっ歯類の脳スライスは、電気生理学、顕微鏡法、薬理学を用いた単細胞分解能を用いた神経回路の組織と機能に関する洞察を得るための、難解な実験的アプローチを提供します。しかし、 インビトロ 実験の設計における大きな考慮事項は、異なるスライス調製物が 、生体内で 観察される神経活動の自然主義的パターンをどの程度再現するかを示す。無傷の脳では、海馬ネットワークは、覚めの完了状態または非レム睡眠中に起こる鋭波波性複合体(SRS)によって例示されるように、動物の行動状態を反映する高度に同期した集団活動を生成する。SRSおよび他の形態のネットワーク活動は、適切な条件下で孤立した海馬スライス中に自発的に出現する可能性がある。強力な脳スライスツールキットを海馬ネットワーク活動の調査に適用するためには、組織の健康と海馬ネットワーク内の機能的な接続性の維持を最適化するアプローチを利用する必要があります。マウスは、経皮的に冷たいショ糖ベースの人工脳脊髄液と浸透する。海馬を含む水平スライスは、シナプス接続性を維持するために450 μmの厚さで切断されます。スライスはインターフェイススタイルのチャンバで回復し、記録のために水没した部屋に移される。記録室はスライスの酸素化を改善するために高流量の人工脳脊髄液の二重表面の重ね合たちのために設計されている。このプロトコルは、複雑で自発的なネットワーク活動の調査に適した健康な組織 をvitroで生成します。

ここでは、このプロトコルで説明したように準備されたHECスライスからの代表的な録音を示します。インターフェース保持室(図1C)での回復後、スライスは水没した記録室に個別に移される(図2B)。記録チャンバには、蠕動ポンプを用いてカルボゲン飽和ACSFが供給される(図2A)。ポンプは最初に保持ビーカーから熱い貯蔵所にACS...

Watch this Scientific Journal Video about Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity at JoVE.com

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

このプロトコルは、自発的な鋭波波リップル活性を示すマウスからの水平海馬内皮質(HEC)スライスの調製を記述する。スライスは、簡略化されたインターフェース保持室でインキュベートされ、記録は、組織の酸素化とネットワークレベルの活動の自発的な出現を促進するために、流れの速い人工脳脊髄液と水没条件下で行われます。

急性マウス脳スライスによる、海馬ネットワーク活動の調査

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

acute-mouse-brain-slicing-to-investigate-spontaneous-hippocampal

Research

JoVE Journal

Neuroscience

8.9K ビュー.  Columbia University. このプロトコルは、自発的な鋭波波リップル活性を示すマウスからの水平海馬内皮質(HEC)スライスの調製を記述する。スライスは、簡略化されたインターフェース保持室でインキュベートされ、記録は、組織の酸素化とネットワークレベルの活動の自発的な出現を促進するために、流れの速い人工脳脊髄液と水没条件下で行われます。

ビデオ: Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

神経科学

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

軽度の外傷性脳損傷後の海馬回路機能の改変に関する調査

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

地中マイクロ電極アレイと非折り畳み海馬準備神経活動の伝播

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

記録と電極アレイに結合された齧歯動物の脳スライスにおけるてんかん活動の変調

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

マウス脳の水平海馬スライス

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

成体マウスのデンテート・ジラスにおける全細胞記録と神経再建のための、逆海馬からの急性スライスの作製

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Source: Whitebirch, A. C. <a href="https://app.jove.com/v/61704">Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity.</a> J. Vis. Exp. (2020)
The video demonstrates a procedure to record the local field potential (LFP) in mouse hippocampal-entorhinal cortex slices. Electrical stimulation is provided at the CA3 stratum radiatum of the hippocampus, which causes changes in the postsynaptic potential at the CA1 stratum pyramidale. The combined change in the membrane potential of CA1 neurons, termed the local field potential, is recorded.

マウス海馬-内膜皮質スライスにおける局所電界電位の記録

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Transcardial perfusion

	Suspend ...