ペドロバリーデラマーサ財団は、この作業を支持した。

ロテノンの1）胃内投与<ol><li>動物を秤量する。 </li><li>必要なロテノン/車両溶液の総容量を計算します。動物の重量のグラムあたりこの場合は0.01ミリリットル。 </li><li>以前に計算されたロテノン溶液の体積（0.625 mg / mLのロテノン、4％カルボキシメチルセルロースおよび1.25％のクロロホルム）または車両の溶液（4％カルボキシメチルセルロースおよび1.25％のクロロホルム）で1 mLシリンジを充電してください。このケースでは、5 mg / kgのロテノンの投与量に相当する。 </li><li>マウスをピックアップし、片手でマウスの尾を持って、他の手の最初の二つの指でそれを引いて、首の皮膚を伸ばす。 </li><li>ヘッドは2本の指で固定されると、手のひらに対してそれを傾くと逆さまそれを回す。第四および第五の指を使って尾を修正。 </li><li>背面に向かって頭にしておくと、マウスの口を開くために口蓋を押してください。ゆっくりと口の中で強制経口投与を紹介。強制経口投与針ではなく、通常の針を使用することが重要です。ゆっくりと、十分胃の方向に強制経口投与を移動する。これは、肺になっ物質を避けることができます。 </li><li>優しく塞栓を押して、ロテノンのソリューションや車両を管理する。完了すると、徐々に強制経口投与を抽出する。 </li><li> oneケージからすべての動物が終了するまで戻って別のケージで動物を置きます。 </li></ol> 2）免疫染色の手順<ol><li>治療後、マウスは、ケタミンは100 mg / kgウントXylazin 10 mg / kgをipおよびintracardially PBS中4％パラホルムアルデヒドで灌流を使用して麻酔する。 </li><li>ガッツはその後、解剖によって抽出され、一晩15％ショ糖に配置されます。翌日、組織が30％スクロースに転送し、4で再び一晩インキュベート℃に</li><li>組織は、組織- TEKに配置し、使用するまで-80℃の冷凍庫に保存されます。 </li><li>クライオスタットを用いて、20ミクロンの小腸の断面積はSuperfrostスライドに転送されます。 </li><li>抗βIII-チューブリンと抗α-シヌクレイン抗体を使用してプロシージャをブロックして免疫染色することはその後、前述の9として実行されます。 </li></ol> 3）α-シヌクレインのインクルージョン画像解析それは画像の起源を知らない外部の人が分析を行うことをお勧めします。したがって、データが体系化されるべきである。画像解析は、フィジーのソフトウェアを用いて行った。 <ol><li>共焦点画像ファイルを開きます。それは各チャネル独立に動作させることが可能になるようにし、チャネルを分割する。 </li><li>解析]を使用して選択する&gt;スケールを設定し、1ピクセルサイズを設定するつもりはないそれらのチャネルを閉じます。 0.13（これは使用目的や解像度に依存します）に既知の距離を設定し、グローバルをクリックしてください。画像のサイズは現在、ミクロン単位で表示されます。 </li><li> α-シヌクレインチャンネルを選択して、神経節を選択し、選択はすべてのスライスに神経節を囲むことを確認してください。 </li><li>プレス画像&gt;画像を複製する重複。神経節を選択した状態で、画像のみが神経節の大きさが複製されます。 </li><li>編集&gt;クリア外側を押してください。選択された領域の外側で画像の一部が黒になります。 </li><li>手順が行ったどれだけ後の手順で決定するイメージを再度複製する。バックグラウンド比のノイズへの高い信号を持っている画像は、画像解析には使用しないでください。 </li><li>イメージ&gt;調整&gt;しきい値を選択し、[MaxEntropy]を選択します。その後の手順がうまくいったことを保証するためにトリミングされた画像と元のスライス上に移動。エントロピーのしきい値は、輝度信号レベルの画像のヒストグラムに基づいており、この閾値よりも高い強度を持つピクセルだけが黒の画像の上に白で表示されますを選択します。 </li><li>プロセスを選択して、滑らかにします。このステップでは、滑らかなエッジの画像のpixeledエッジを変換します。 </li><li>プロセスを選択し、[バイナリ]をクリックし、バイナリを作成]を選択します。画像は白画像上に黒として表示されます。 </li><li>重複した画像上で次のステップを実行するためにイメージを複製する。 </li><li>解析&gt;粒子を分析をクリックしてください。 [表示]を選択します：アウトラインをし、集計]をクリックしてください。残りの設定は、デフォルトで葉のはずです。この関数は、信号と一緒にグループ化されているものを持つピクセルの合計数を認識します。信号に加えてピクセルのグループの数と平均サイズと画素の全表面が報告されています。 </li><li>うまく調整する最大のスライスを比較して選択します。スライスがマークされます。 </li><li>トータルα-シヌクレインの表面、介在物の数と、平均粒径を持つデータテーブルをコピーします。他のExcelスプレッドシートまたは注釈付きにデータを貼り付けます。 </li><li>フィジーに戻るとβ-チューブリンのチャンネルで以前に選択したスライスを見つけ、そしてそれを選択します。 </li><li>神経節の領域を選択して、測定を選択し、[分析]をクリックします。別のテーブルが表示されます神経節の全表面。 </li><li> α-シヌクレインの測定の近くにExcelシートにテーブルからデータを抽出したり、他の場所にそれをコピーします。 </li><li>エクセルを使用して、総α-シヌクレインの表面と神経節の大きさで正規化された包接数を取得するために神経節の表面からα-シヌクレインの測定で得られたさまざまなパラメータを分割。 </li></ol> 4）代表者の結果強制経口投与プロトコルが正しく実行されると動物のための不便は最小限です。ロテノンのこの濃度を使って治療は、血液中のロテノン濃度なしENS上ロテノンの局所効果を可能にし、筋肉や脳ミトコンドリア複合体の阻害、私も治療の1.5ヶ月（図1参照）の後。画像解析が正しく行われている場合、α-シヌクレインのパターンの厳密な分析が可能なはずです。それは、α-シヌクレイン（合計面）、α-シヌクレインのセルのパターン（封入体の数）とα-シヌクレイン封入体の存在（介在物寸法）（図2）の合計額に信頼性の高いデータを提供します。 <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="図1" /> 図1。ロテノンの運動機能障害は、血液や中枢神経系におけるロテノンの検出なしでマウスを処理した。 、標準（50 ng / mL）を、20、10および5 mg / kgを投与したマウスの脳試料からのクロマトグラム。BとC、ロテノンの血中濃度（B）およびCNS（C）の定量化。B、血中レベル1 、治療後の2と3時間。マウスは3つのグループ（N = 3）に分割し、2.5、5、10、20 mg / kgのロテノン（N = 3）で処理した、血液300μLのはロテノン投与後1、2、3時間を抽出し、一緒にプールしたHPLC分析。C、マウスは5（N = 3）、10（N = 3）および20（N = 1）mg / kgのロテノン、脳や脳幹で一日一回一週間の治療を受けていた1と2時間後に抽出した最後の管理およびHPLC分析。D、1.5カ月治療したマウスの筋肉および脳のサンプル中のミトコンドリア複合体I活性のために準備。 <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="図2" /> 図2。ローカルで管理さロテノンは、ENS神経節における神経膠症とα-シヌクレインのリン酸化、蓄積して凝集を誘導する。（スケールバーは20μm）。、B、C、抗βIII-チューブリン、α-シヌクレインと十二指腸のDAPI染色（B）と回腸（は、C）のセクション。 3ヶ月のコントロール（）と比較するとBの矢印は、1.5ヶ月の治療は、腸神経系の神経節内の増加、α-シヌクレイン斑点状のパターンを誘発した。 Cの矢印、3ヶ月の治療は、より大きなα-シヌクレインのインクルージョン（ø&gt; 6μm）の形成を誘導した。D、抗α-シヌクレイン、チオフラビンSとDAPIを用いて免疫蛍光染色。 Dの矢印、わずか3ヵ月投与したマウスは、これらの大規模なα-シヌクレインの蓄積の凝集を示した。E、E&#39;、E &#39;&#39;、画像解析の定量化の手順。E、ENSの神経節を示す定量化に使用される画像の例は、示すαシヌクレインに対する免疫染色別のインクルージョン。E&#39;、画像解析のプロトコルの後に得られた画像の例。E &#39;&#39;、オリジナルのインクルージョン（緑）とプロトコル（黄色の線）を実行後に得られる表現の一部との間のオーバーラップ。FG、に示すように実験の定量化ACは、自動セグメンテーションとエントロピーベースのスレッシュホールド法を用いて行った。シングルアスタリスク、P &lt;0.05、および二重アスタリスク、P &lt;0.01。F、各列が総α-シヌクレインの面/神経節面を表す。G、各列には、α-シヌクレイン封入体/神経節面の数の合計を表します。すべてのグラフは、平均± SEMを示す

<ol>
	<li>Braak, H., de Vos, R. A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. 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フランシスコパン- Montojoは、この動物モデル（出願番号PCT / EP 2009/005688）の保留中の特許出願しています。

胃内投与は、以前は12を行われている。しかし、印伝の原稿によると、ロテノンは、0.5％カルボキシメチルセルロースのナトリウム塩（CMSS）単独で溶解投与した。ロテノンは高いliphophylic物質です。したがって、ロテノンは、単独で0.5％のCMSSに溶解することができないので、行えば沈殿する。クロロホルムを使用すると、沈殿を避ける均等に分散さロテノン懸濁液を作成します。ま?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
</tr>
<tr>
<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
</tr>
<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
</tr>
<tr>
<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FIJI</td>
<td>&nbsp;</td>
<td>http://pacific.mpi-cbg.de</td>
<td>&nbsp;</td>
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oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

パーキンソン病（PD）患者では、関連する病理学は、腸神経系（ENS）1,2、嗅球（OB）、迷走神経背側運動核（DMV）3、中間外側とりわけ関与する特徴的なパターンに従います。脊髄の4と黒質の核、4,5病の神経病理学的ステージングの基礎を提供する。 ENSとOBが最も露出神経の構造と影響を受ける最初のものです。興味深いことに、PDは、農薬暴露6-8関係している。ここでは詳細に私たちの以前の研究9で使用される2つの方法を示しています。 ENSで局所的に作用するロテノンの影響を分析するために、我々は1歳C57/BL6マウスに強制経口投与を使用してロテノンを投与。ロテノンは強くミトコンドリア複合体のI 10を阻害する広く使用されている農薬です。それは非常に親油性と胃腸管11に吸収される。我々の結果は、ロテノンの5 mg / kgの投与は、筋肉や脳内のミトコンドリア複合体Iの活性を阻害しなかったこと。示したしたがって、我々の投与方法のロテノンを使用すると、肝門脈のシステムを横断しておらず、もっぱらENSに作用していたことを示唆している。ここでは、ENSにおけるα-シヌクレインの蓄積で、農薬の効果を分析するために使用される強制経口投与し、画像解析のプロトコルを使用して農薬を管理する方法を示しています。最初の部分は、目的の正確な濃度で殺虫剤（ロテノン）の胃内投与を可能にする方法を示しています。第二の方法は、画像解析ソフトウェアを使用してENSにα-シヌクレインの蓄積を分析するために半自動画像解析のプロトコルを示しています。

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

パーキンソン病は、農薬への暴露に関係している。ここでは、所望の濃度と腸神経系ではα-シヌクレインの蓄積で、その効果を分析する方法で胃管を使って殺虫剤を提供する方法を示しています。

腸神経系ではα-シヌクレインの介在物の強制経口および画像解析を用いたロテノンの経口投与

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

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Neuroscience

16.8K ビュー.  Technische Universität Dresden. パーキンソン病は、農薬への暴露に関係している。ここでは、所望の濃度と腸神経系ではα-シヌクレインの蓄積で、その効果を分析する方法で胃管を使って殺虫剤を提供する方法を示しています。

ビデオ: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

神経科学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

腸神経系のその場のCa 2+イメージングに

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

パーキンソン脳からの可溶性および不溶性のα-シヌクレインの逐次抽出

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

α-シヌクレイン線維の末梢接種後のトランスジェニックマウスにおける神経炎症の生物発光イメージング

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

メソッドおよびアデノ随伴ウイルス ラットの静脈内投与と中枢神経系の伝達の評価のためのヒント

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

線維形成の強力な細胞モデルとして α-シヌクレインのミクロソーム関連付けられた集計一次ニューロンの外因性投与

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

化学遺伝子解析で作られた皮質間ニューロン前駆体を早期産後マウス脳に移植

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

モノマーからのαシヌクレイン予備型フィブリルの生成と生体内での使用

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...