NIHの助成金AG027297、ケンタッキー州脊髄と頭部外傷の研究のトラストからの賞を受賞、とクレバーグ財団からの贈り物でサポートされて働く。

<ol>
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このビデオ記事の生産は、ライカマイクロシステムズが後援した。

このプロトコルで説明する手順は、脳の解剖は、若い成体ラットのように、熟成に最低でも迅速かつ効率的に行われることを保証するのに役立ちます。我々はまた、LTPとLTDに独自のスライスの調査をセットアップする初心者のために十分な詳細を提供します。高齢化とシナプス機能と可塑性の広告の変更の更なる探査が目標の一つである場合は、さらに検討に値する上記alluded少なくとも2つの他の方法論上の問題は、、そこです。最初に、いくつかのラボは、Ca 2ことが示されている+：録音ACSFでのMg2 +比は、海馬スライス2,10,24,25で誘導シナプス可塑性に著しい影響を持つことができます。哺乳類の脳脊髄液は、Ca 2 +に：Mg2 +の比は約1（例えば26を参照）です。しかし、ACSF のCa 2 +：Mg2 +の比に近い2〜一般的にシナプス機能と可塑性のスライスの研究に使用されています。初期の研究では、この方法はおそらく、その後、すべての可塑性の研究のためのルーチンになってから、LTPの誘導を最適化するために適応されました。しかし、この習慣が原因の神経のCa 2 +調節でよく特徴の違いの老化とADの研究で問題となる可能性があります。具体的には、Ca 2 +流入及び/またはCa 2 +誘発Ca 2 +放出は、神経細胞活性化3,27-31時の老齢ラットおよび/ ​​またはADのモデルマウスで上昇している。 LTDの誘導は、ACSFのCa 2 +濃度の微妙な変化に特に敏感である。 2〜Mg2 +の比が近い、としている成人で堅牢な会社を見てきた：Ca 2 +を用いた研究は、一方2mMのカルシウムの+と2mMのMg 2 +を、一般的に歳のための株式会社の結果ではなく、若い成人の動物2を 、使用している我々のプロトコル、 32老齢ラットで低下LTDと一緒に年齢の差が2,10またはの不在。これらの観察は慎重にACSFのCa 2 +とMg2 +のレベル高齢者と若年成人の動物では、Ca 2 +依存性の可塑性を比較する際に考慮する必要性を強調。 第二の方法論的問題は、シナプス強度のシナプス後脱分極33と可能老化/遺伝子型の違いでLTPの強い依存性を懸念。で典型的なLTPの実験、ベースラインとLTPの刺激強度は、一般的に半最大（または三四半期最大）EPSPの振幅を生成するために調整されます。潜在的な問題は、老齢ラットとAPP/PS1マウスには、通常、ベースラインEPSPの値も老齢ラットとAPP/PS1マウスで小さくなることを意味し、彼らの若いおよび/または野生型の対応への相対的なシナプス強度の低下を示すということです。小さ ​​いEPSPのは、LTP 33を誘導するための低下の確率で、その結果、LTPの刺激の間に以下の脱分極に変換する場合があります。このため潜在的な混同するから、それはこれらの動物は、スループットの赤字、可塑性の欠損または両方を示すかどうかを決定することは困難である。つまり、高齢者および/またはAPP/PS1マウスにおけるLTP誘導メカニズムは、（無可塑性の赤字）機能的に無傷かもしれませんが、十分にこれらの条件下で（スループットの赤字）刺激。スループットと可塑性のメカニズムのためのメカニズムは、特定の薬理学的治療に非常に異なった反応を可能性があるため、この区別は、非常に重要です。我々は、前のLTPの刺激へのすべてのスライスに同じレベル（例えば1 mVの）にEPSPの振幅を正規化することによってLTP誘導に対するスループットが低下の影響を最小限に抑えるようにしてください。他の戦略は（LTPの刺激の間にグループ間での膜電位を等しくする電圧または電流クランプの例を使用）だけでなく、効果的かもしれない、とこれらの動物モデルでLTPを調査する際に考慮する必要があります。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> NaClの</td><td>フィッシャー</td><td> BP358 - 1 </td></tr><tr><td>塩化カリウム</td><td>フィッシャー</td><td> BP366 - 500 </td></tr><tr><td> KH 2 PO 4（一塩基） </td><td>シグマ</td><td> P5379 - 100G </td></tr><tr><td> MgSO 4を </td><td>シグマ</td><td> M2643 - 500G </td></tr><tr><td>のCaCl 2（二水和物） </td><td>シグマ</td><td> C3306 - 250G </td></tr><tr><td> NaHCO 3の </td><td>フィッシャー</td><td> S233 - 500 </td></tr><tr><td> C 6 H 12 O 6（ブドウ糖） </td><td>フィッシャー</td><td> BP350 - 1 </td></tr></tbody></table> 表1。必要な試薬<table border="1"><tbody><tr><td> 機器の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>三角フラスコ</td><td>フィッシャー</td><td> FB - 500 - 2000 FB - 500 - 1000 </td><td></td></tr><tr><td>水族館バブラー</td><td></td><td></td><td>オキシメディアのために使用。ほとんどのペット店で購入できます</td></tr><tr><td> 50mLのガラスビーカー</td><td>フィッシャー</td><td> 02 - 540G </td><td> ACSFで脳storarge用</td></tr><tr><td>パラフィルム</td><td>フィッシャー</td><td> 13-374-10 </td><td></td></tr><tr><td>小動物のギロチン</td><td>世界精密機器（WPI） </td><td> DCAP - M </td><td></td></tr><tr><td>平らな紙タオル</td><td></td><td></td><td></td></tr><tr><td> ＃11フェザー外科ブレード</td><td>フィッシャー</td><td> 08から916 - 5B </td><td></td></tr><tr><td>ビービー骨はさみ</td><td>ファイン科学ツール（FST） </td><td> 16044〜10 </td><td></td></tr><tr><td> Lempert Rongeurs </td><td> Roboz </td><td> RS - 8321 </td><td>ラットのために使用します。 </td></tr><tr><td>フリードマン-ピアソンRongeurs </td><td> FST </td><td> 16020〜14 </td><td>マウスやラットのために使用します。 </td></tr><tr><td>海馬のツール</td><td> FST </td><td> 10099〜15 </td><td></td></tr><tr><td>スプーン</td><td></td><td></td><td>プラスチック製のティースプーンが行います</td></tr><tr><td>へら</td><td>フィッシャー</td><td> 21から401 - 25A </td><td>へら</td></tr><tr><td>外科的虹彩鋏</td><td> FST </td><td> 14058〜09 </td><td></td></tr><tr><td>プラスチック製のトランスファーピペット</td><td>フィッシャー</td><td> 13-711-43 </td><td></td></tr><tr><td> 110ミリメートルワットマンろ紙</td><td>フィッシャー</td><td> 09 - 805E </td><td>ワットマンの猫。 1001-110 </td></tr><tr><td>ガラスのペトリ皿</td><td>フィッシャー</td><td></td><td></td></tr><tr><td>ミクロトームを振動ライカVT1000Pマニュアル</td><td>ビブラトーム</td><td></td><td></td></tr><tr><td> 0.1ミリメートルFA - 10フェザーSブレード</td><td>テッドペラ</td><td> 121から9 </td><td> 0.1ミリメートルFA - 10フェザーSブレード</td></tr><tr><td>ホウケイ酸ガラスパスツールピペット（ゴム球付き） </td><td>フィッシャー</td><td> 13から678 - 20A </td><td>大きな開口部のためにヒントが破損していると熱研磨：スライスを転送するための</td></tr><tr><td> 35ミリメートルPolysterine培養皿</td><td>コー​​ニング</td><td> 430588 </td><td>解剖後にスライスを収集するために使用</td></tr></tbody></table> 表2。解剖のためのツールと材料<table border="1"><tbody><tr><td> 装置/材料 </td><td> 会社 </td><td> コメント（省略可能） </td></tr><tr><td>保持チャンバー</td><td>カスタムは、内蔵</td><td></td></tr><tr><td> P - 97水平ピペットプラー</td><td>サター器械（株） </td><td></td></tr><tr><td>防振テーブル</td><td>テクニカルマニュファクチャリング株式会社（TMC） </td><td></td></tr><tr><td>ファラデー箱</td><td>カスタムの構築</td><td></td></tr><tr><td>ボトムサイドアーム付きパイレックスアスピレータボトル（製品番号1220 - 1L） </td><td>コー​​ニング</td><td></td></tr><tr><td>レギュレータと重力contolled IVセット（製品＃2C8891） </td><td>バクスター</td><td></td></tr><tr><td>中央の真空ライン</td><td></td><td>最も近代的なラボで利用できる</td></tr><tr><td> 95％O 2 / 5％CO 2ガスのミックス</td><td>スコットT -グロス（株） </td><td></td></tr><tr><td> TygonTMラボチューブ O 2 / CO 2の配信のための</td><td>フィッシャーサイエンティフィック</td><td>非毒性、非酸化性、さまざまなサイズで提供されます。 </td></tr><tr><td> EclipseのE600FN顕微鏡</td><td>ニコン</td><td> infaredフィルタの近くに、10倍および40倍の目標とし、GFP、DS - Red2フィルター</td></tr><tr><td>クールなスナップESデジタルカメラ</td><td> Photometrics </td><td>クールなスナップESデジタルカメラ</td></tr><tr><td> X - Citeの蛍光灯照明装置</td><td> EXFO </td><td> X - Citeの蛍光灯照明装置</td></tr><tr><td>顕微鏡プラットフォーム</td><td>シスキユー</td><td>カスタムは、組み立て</td></tr><tr><td> RC - 22水中録音室（製品番号64から0228まで） </td><td>ワーナーインスツルメンツ（WI） </td><td> P - 1プラットフォームとステージアダプタ（ワーナーから製品＃64〜0277）が必要です</td></tr><tr><td> TC2BIP 2/3Ch温度コントローラ </td><td>セルMicrocontrols </td><td> TC2BIP 2/3Ch温度コントローラ </td></tr><tr><td> 4軸手動ミニチュアマニピュレータ</td><td>シスキユー</td><td></td></tr><tr><td>プラチナイリジウムワイヤー（0.002で） （項目＃PTT0203） </td><td> WPI </td><td></td></tr><tr><td> A365刺激アイソレータ</td><td> WPI </td><td> A365刺激アイソレータ</td></tr><tr><td> 700BアンプをMulticlamp </td><td>アクソンインスツルメンツ</td><td></td></tr><tr><td> Digidata 1322A / Dコンバータ</td><td>アクソンインスツルメンツ</td><td></td></tr><tr><td>ソフトウェアをPClamp </td><td>アクソンインスツルメンツ</td><td></td></tr><tr><td>パーソナルコンピュータ（Pentium 4プロセッサ） </td><td>デル</td><td></td></tr></tbody></table> 表3。電気生理学的機器と材料

preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology

げっ歯類の海馬スライス標本では、おそらく哺乳類のシナプス機能と可塑性を調査するための最も広く使用されるツールです。海馬は、酸素、人工脳脊髄液中の時間のために実行可能なまま、ラットやマウスとスライスから迅速かつ容易に抽出することができます。また、基本的なelectrophysisologicテクニックは簡単に海馬スライスにおけるシナプス機能の研究に適用され、認知障害のための最適なバイオマーカーのいくつかを提供している。海馬スライスは、学習や記憶に関与するシナプス可塑性のメカニズムの研究のために特に人気があります。海馬スライス（またはその欠如）のシナプス伝達効率の長期増強と抑うつ（LTPとLTD）の誘導の変化が頻繁に認知障害のある動物の神経学的表現型を記述すると/またはの作用機序を評価するために使用されます向知性化合物。この記事では、我々はラットおよび脳老化とアルツハイマー病（AD）1-3に関連付けられているシナプスの変化の研究のためのトランスジェニックマウスから海馬切片を調製するために使用する手順の概要を示します。老齢ラットとADのモデルマウスの使用は、その研究に若いラット及び/またはマウスを使うことに慣れ研究者への挑戦の固有のセットを提示することができます。老齢ラットでは、厚い頭蓋骨と脳の抽出及び/または解剖を遅らせ、その結果、シナプス機能と可塑性の本当の年齢の違いを無効にするか、または誇張することができます若いラットとマウスに、より厳しい結合組織を持っている。老化とアミロイド病理がまた再び生理的な評価から引き出された推論を複雑にして、解剖の手順の間に持続的な海馬の損傷を悪化させる可能性があります。ここで、我々はこれらの問題を最小限に抑えるために解剖の手順の実行中に実行する手順を説明します。 &quot;健康&quot;とラットやマウスからの&quot;不健康&quot;のスライスに取得したシナプス応答の例も同様に代表的なシナプス可塑性の実験として、提供されています。これらの動物モデルにおけるシナプス機能（例えば、記録のソリューションコンポーネント、刺激パラメータ）上の他の方法論的な要因の影響の可能性についても議論されています。この記事の焦点は老齢ラット、トランスジェニックマウスの使用上にある間、生理をスライスする初心者は、齧歯類モデルのさまざまな方法を使って、自分の研究をはじめるためにはここに十分な詳細を見つける必要があります。

Watch this Scientific Journal Video about Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology at JoVE.com

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

この記事では、ラットと脳の老化とアルツハイマー病などの加齢に伴う神経変性疾患、関連付けられているシナプスの変化の研究のためのトランスジェニックマウスから海馬切片を調製するための手順の概要を説明します。

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 ...

preparation-acute-hippocampal-slices-from-rats-transgenic-mice-for

Research

JoVE Journal

Neuroscience

93.7K ビュー.  University of Kentucky College of Public Health. この記事では、ラットと脳の老化とアルツハイマー病などの加齢に伴う神経変性疾患、関連付けられているシナプスの変化の研究のためのトランスジェニックマウスから海馬切片を調製するための手順の概要を説明します。

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

神経科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

マウス海馬にオリゴマーアミロイドベータの定位注入

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

げっ歯類からの急性海馬スライスを用いて、シナプスタグ付け/キャプチャとクロス捕獲調査

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

少量循環、血流 - と水没型チャンバー システムで管理急性海馬スライスにおけるシナプス可塑性を記録

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常マウスおよびハンチントンマウスからの急性脳スライスにおけるグルタミン酸放出および取り込みの単一シナプス指標

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

電気生理学的記録のための急性ヒト海馬スライスの調製

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...