この作品は、裾には、NIHの助成金DA15169およびDA035224によって賄われていた

全ての動物の取り扱いと組織の収穫は、承認されたプロトコール番号のA1506（Melikian、PI）以下、マサチューセッツ大学医学部動物実験使用委員会（IACUC）大学のガイドラインに従って行われた。 必要な溶液 人工脳脊髄液（ACSF） -新鮮な毎日を作る 125mMの塩化ナトリウム、2.5mMのKCl、1.2mMののNaH 2 PO 4、1.2のMgCl 2、2.4のCaCl 2、26 mMの炭酸水素ナトリウム、および11 mMグルコース 注： 炭酸水素ナトリウムとグルコースを除いた、10倍の原液としてACSFを準備します。新鮮なNaHCO 3およびグルコースを補う、10倍のストックから毎日1X使用液を作る。 ショ糖を補充ACSF（SACSF） -新鮮な毎日を作る 250 mMの成功、2.5mMのKCl、1.2mMののNaH 2 PO 4、1.2のMgCl 2、2.4のCaCl 2、26 mMの炭酸水素ナトリウム、および11 mMグルコースローズ 注： 炭酸水素ナトリウムとグルコースを除いた、10倍の原液としてSACSFを準備します。新鮮なNaHCO 3およびグルコースを補う、10倍のストックから毎日1X使用液を作る。 スルホ-N-ヒドロキシスクシニル-SS-ビオチン （スルホ-NHS-SS-ビオチン、ピアース·ケミカル·カンパニー） 原液をDMSO中200 mg / mlのも、複数回の凍結/融解サイクルに耐性がある必要があります。アリコートを-20℃で保存するサクシニルエステルは急速に水溶液中で加水分解されるので、作業溶液は、前のスライスに適用するにすぐに準備する必要があります。 スライスクエンチソリューション 100 mMグリシンを補給しACSF RIPA溶解BuffeR 10mMのトリス、pH7.4、150mMのNaCl、1.0mMのEDTA、1％トリトン-X-100、0.1％SDS、1％デオキシコール酸ナトリウム プロテアーゼ阻害剤（RIPA / PI）でRIPA -新鮮な毎日を作る RIPAは、1μMロイペプチン、1μMペプスタチン、1μMアプロチニン、および1mMフェニルメチルスルホニルフルオを補った。 1。脳スライスを準備します<ol><li>作る新鮮な1X SACSFおよび1X ACSF </li><li>小さなビーカーに氷の上SACSFを冷やす。これは、新たに採取したマウスの脳を保持するために使用される。 </li><li> 95％/ 10％のO 2 / CO 2、氷上で20分でバブリングして酸素とACSFとSACSFを飽和さ。 </li><li> P30-38マウスは、最適な組織の生存のために使用されるべきである。頸椎脱臼や断頭により動物を犠牲にし、急速に予備冷却、酸素化SACSFに脳を削除します。 </li><li>振動ミクロトームを用いて、関心領域内の300μmの脳切片を作る。 </li><li>必要に応じて、スライスはさらにそれぞれ、対照および実験スライスとして使用するために、特定の脳領域または別々の左右の半球を濃縮する前に回復を解剖することができます。 </li><li>熱加工パスツールピペットを用いて、24ウェルプレートにセットチャンバ底メッシュのスライスを転送する。 </li><li>スライスが継続的、穏やかな泡立ちで、酸素化ACSF中で、40分間31℃を回復することができます。 </li></ol> 2。薬物治療（該当する場合）とスライスビオチン化<ol><li>回収に続いて、スライスを3回洗浄する予め温めた（37℃）で、酸素化ACSF 95％/ 5％O 2 / CO 2でバブリング絶えず。 </li><li>試験化合物を追加し、継続的な酸素化でインキュベートする。 <ol><li>便宜のために、10倍濃縮された薬剤の1/10の容量を追加して、静かに転倒混和。 </li><li>所望の温度の水浴中で穏やかにプレートを振る。 </li></ol></li><li>次の薬物治療、急速に氷冷たいACSFで3回洗浄することにより、スライスを冷やす。 </li></ol> 3。ビオチン表面タンパク質<ol><li>直前のラベルに、氷冷​​ACSFでの準備1.0 mg / mlのスルホ-NHS-SS-ビオチン。 </li><li>スライスに0.75ミリリットルスルホ-NHS-SS-ビオチンを加え、氷上で45分のスライスをインキュベートする。 </li><li>氷上の氷冷ACSF中で10分間インキュベートして、氷冷ACSFで迅速にスライスを3回洗浄します。 </li><li>氷冷たいスライスクエンチバッファーでスライス3回洗浄し、無料のスルホ-NHS-SS-ビオチンを急冷し、氷上で、0.75ミリリットルスライスクエンチ緩衝液で2回、25分間インキュベートする。 </li></ol> 4。組織溶解物を準備します<ol><li>氷冷ACSF内のスライス3回洗浄し、ファイアーポリッシュパスツールピペットを用いてマイクロ遠心チューブに各スライスを転送する。 </li><li> 200 XG、1分、慎重に吸引残りのACSFを遠心分離することにより穏やかにペレットをスライス。 </li><li> P200ピップを通して一度ダウンして400μLの氷冷RIPA / PIを加え、ピペッティングにより組織を破壊し、ディスケット。 </li><li>転写を新しいチューブにスライス/ RIPA解離し、溶解を完了するために、回転、30分間、4℃でインキュベートする。 </li><li>遠心分離して細胞破片をペレット化し、18,000×gで、15分間、4℃ </li><li>標準としてウシ血清アルブミン（BSA）を用いて、BCAタンパク質アッセイを用いて溶解物のタンパク質濃度を決定する。 </li></ol> 5。ビオチン化タンパク質を単離<ol><li>ビーズ/総タンパク質比を最適化します。 注：個々のタンパク質発現レベルは、異なる脳領域を横切って広く変えることができる。組織溶解物の所与の量のビオチン化タンパク質の全てが捕捉されない限り、正確表面発現の潜在的な変化を検出することができない。したがって、経験的に前の新しいスライスビオチン化研究に着手する特定のタンパク質/脳領域のための最適なビーズ/総タンパク質比を決定することが不可欠である。 <ol><li>増加量が25μlのストレプトアビジンアガロースビーズをインキュベートその後、組織溶解液（おおよその範囲25〜200μgの）のS、および結合、洗浄および溶出工程のために以下に記載するように進んでください。 </li><li>得られた免疫反応性のバンドを定量化し、その結合の線形範囲内のビーズ/溶解物比を選択すると、増加またはタンパク質の表面発現の減少のいずれかの正確な定量化を可能にする。 </li></ol></li><li>ストレプトアビジン - アガロースビーズを準備します<ol><li>すべてのサンプルに必要な総ビーズ体積を決定します。 25μL/サンプル= 100μlのビーズ+ 1余分= 125μlのビーズで、その量に十分なビーズ容量プラス1の余分な（ つまり 4サンプルを準備します。 </li><li>渦ストレプトアビジンビーズストックピペットから所望のボリューム。 P200ピペットを使用している場合は、閉塞またはビーズの損傷を防ぐために、チップの先端を切り落とした。 </li><li>部屋の気性で、18,000×gで1分間遠心分離することにより保存料、各RIPA添加後ボルテックスと洗浄の間に収集ビーズを除去するために0.5〜1.0ミリリットルRIPA / PIでビーズを3回洗浄しますature。 </li><li>真空フラスコに付着したガラスパスツールピペットを用いて洗浄の間にバッファをオフに吸引除去する。吸引時にガラスパスツールピペットの先端に取り付けられたプラスチックのP200チップはより細かい制御を提供します。それがピペットにビーズを吸引するリスクなどには、最初の2回の洗浄のために絶対的に全てのバッファを削除する必要はありません。最終洗浄後、ビーズを吸引することなく、可能な限り余分なバッファを削除します。 </li><li>再懸濁するために上下にピペッティングして、戻って、元のボリュームにビーズを持って来るRIPA / PIを追加します。ビーズが管壁に固執するように、この時点では、ボルテ​​ックスを避けてください。 </li><li>カットオフチップでP200を使用したマイクロ遠心チューブに一定分量ビーズ。ピペットは、ビーズを確保するために、サンプリングの間に数回上下に均等にチューブの中で懸濁し、分散したまま。 </li></ol></li><li>ストレプトアビジンビーズに結合ビオチン化タンパク質<ol><li>アガロースビーズを含むチューブに細胞溶解物を配布します。実験用のWHE<html>複数のサンプルが比較されているRE、正確な比較のために、各サンプルについて同量のタンパク質を使用してください。 </li><li> 200μlの最小体積をサンプルにもたらすために、追加のRIPA / PIを追加します。これはサンプルがインキュベーション中に十分に混合することを保証し、また、サンプル間でタンパク質濃度を統一。 </li><li>チューブローテーターにチューブをセットし、4℃で一晩混合</li><li>別々のチューブにおいて、各サンプルに対して使用される全ライセート体積と同等のものを分注する。試料容積が高い場合あるいは、総溶解物体積の割合は、SDS-PAGEゲル上で最大負荷容量を収容するために、代わりに予約することができる。これらは、総タンパク量を表面発現を正規化するために使用される。 </li><li>等しい体積の2倍または6×SDS-PAGE試料緩衝液の1/5体積いずれかのサンプルが分析されるまで-20℃でビーズ試料、またはストアと並行して、4℃にてインキュベートのいずれかを追加する。 </li></ol></li></ol>ove_title &quot;&gt; 6。溶出し、サンプルを分析<ol><li>室温で、18,000×gで、2分間遠心分離することにより、ビーズを沈殿。 </li><li>上清を吸引し、洗浄ビーズ0.75ミリリットルRIPAで3回。ビーズの損失を最小限に抑えるには、洗浄の間にビーズ上のバッファの小さなヘッド体積を残す。最後の洗浄後、ビーズペレットを中断することなく、可能な限り多くのRIPAを削除します。 </li><li>ジスルフィド結合を還元することによりストレプトアビジンビーズからのビオチン化タンパク質を溶出する。サンプルバッファーを減らす25μlの2X SDS-PAGEは、ウェルに加え、ボルテックスし、試料を30分間回転させ、室温。 注：多くの膜タンパク質は厳しく、その電気泳動移動度を損なう、SDS-PAGEサンプル緩衝液中で煮沸したときに凝集する傾向が高い。これは調査されたタンパク質の場合である場合には、試料の加熱を回避し、その代わりに、室温で徐々に溶出する。沸騰（並列に評価し、他のタンパク質が沸騰を必要とする場合など ）絶対に必要であるならば、SAMPL電子バッファは、凝集を最小限にするために2 M尿素（最終濃度）を補充することができる。いくつかの場合に凝集を低減するのに有効であるが、尿素はしばしばバンド外観を損なう。 </li><li>イムノブロットによりサンプルを分析する。 </li><li>総溶解物試料を解凍し、ビーズ試料を、30分間、室温と平行に回転する。 </li><li> SDS-PAGEゲル上の別々のタンパク質。 </li><li>免疫ブロッティングにより目的のタンパク質を同定する。 <ol><li>バンドは適切な定量化のための検出の直線範囲内で検出されたことを確信。 </li><li>ビオチン化試薬が破損/侵害の細胞を介した細胞内タンパク質へのアクセスを得ていないことを保証する。これは、最良の調査中の細胞型に特異的な細胞内タンパク質について並行して免疫ブロットにより達成される。 </li><li>バンド密度を定量化し、総タンパク質発現レベルの％として相対タンパク質表面密度を計算する。 </li></ol></li></ol>

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Neurosci. 29, 6794-6808 (2009).</li><li>Rao, A., Simmons, D., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subcellular+distribution+of+endosomal+compartments+and+the+dopamine+transporter+in+dopaminergic+neurons.">Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons.</a> Mol. Cell Neurosci. 46, 148-158 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-752 (2011).</li></ol>

著者らは、この研究と矛盾する財政的利害関係はありません。

脳内での批判的な影響シナプスシグナリングエンドサイトーシスの人身売買は、それが定量的に大人の神経細胞内のタンパク質表面発現の変化を測定することが困難な証明されたことを長年の知識にもかかわらず。本研究では、急性脳スライスにex vivoで表面タンパク質を標識するために信頼性の高いアプローチを報告する。彼らはシナプス結合およびその調製時間後に細胞生存率アッ...

エンドサイトーシスの人身売買は、内在性膜タンパク質の様々な形質膜提示を微調整ユビキタス細胞メカニズムである。エンドサイトーシスは、細胞内環境1に重要な栄養素を提供し、受容体の活性化2に対応して受容体のシグナル伝達を脱感作する。バック形質膜へのエンドサイトーシス再循環はさらに、細胞表面3でのタンパク質の発現レベルを増加させることによって細胞内シグナル伝達を増強することができる。また、膜輸送の摂動は、タンパク質のエンドサイトーシス輸送を支配する分子メカニズムを研究する必要性を強調し、多数の疾患および病的状態に関与している4,5。多くのタンパク質は、古典的なクラスリン依存性内在化機構を利用しながら、過去数年間の証拠をマウントすると、複数のクラスリン非依存エンドサイトーシスの機構が増加、アレイのエンドサイトーシスの可能性を支配することを実証しているタンパク質6,7。従って、生理学的関連システム売買を容易にするエンドサイトーシスの機構を調査する必要性が大幅に高まっている。 脳では、受容体、イオンチャネルや神経伝達物質輸送体のエンドサイトーシスの人身売買は、シナプス可塑8月11日 、最終的には神経細胞の興奮性とシナプス応答に影響を与える虐待12月15日の薬物に対する応答を確立する上で主要な役割を持っています。神経細胞のトラフィッキング研究の大半は、異種発現系または培養一次ニューロンのいずれかに依存して、今日まで、どちらも確実成体ニューロンにおける再生時のメカニズムを反映することができる。ここでは、定量的に成体齧歯類に由来する急性脳スライスにおける表面タンパク質レベルを測定する表面ビオチン化を使用するアプローチを報告する。このアプローチを使用して、我々は、マウスの線条体ドーパミントランスポーターが急速に再内在ことを実証するデータを提示するホルボールエステルにより媒介されるプロテインキナーゼC（PKC）の活性化に応答。

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

エンドサイトーシス調節トラフィッキングを動的分の時間スケール上の受容体、イオンチャネル及びトランスポーターの細胞表面提示を制御することにより、神経調節様々なイベントを容易に中枢機構である。個々のタンパク質のエンドサイトーシス輸送を制御する機構の幅広い多様性があります。売買の分子基盤を調査する研究は、主に定量的に、外因性刺激および遺伝子操作に応答して、膜タンパク質の表面発現の変化を測定する表面ビオチン化に依存してきた。しかし、このアプローチは、主に忠実に、成人ニューロンにおける遊んで生理学的に関連するメカニズムとは異なる場合があり、培養細胞が挙げられるが、これらに限定されています。また、培養細胞のアプローチは、人身売買の仕組みにおける領域特異的な差異を過小評価することができる。ここでは、急性脳スライス標本に細胞表面ビオチン化を拡張する手法を記載している。我々この方法は、成人ニューロンにおける膜タンパク質の表面レベルの急激な変化を測定するために、忠実度の高いアプローチを提供することを実証する。このアプローチは、神経細胞のエンドサイトーシス輸送の分野において広範な有用性を有する可能性がある。

ニューロンのドーパミントランスポーターは、細胞株16-20におけるPKCの活性化に応答して内在化される。細胞株および種々の発現系におけるPKC誘発DAT表面損失を実証する多くの報告にもかかわらず、培養されたドーパミン作動性ニューロン21-23でこの知見を確認するために挑戦してきた。私たちは、直接DATが大人のドーパミン作動性ニューロンにおけるPKCの活性化に応答して?...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

神経細胞の膜輸送を動的に原形質膜タンパク質の可用性と大幅に影響を与える神経伝達を制御します。今日まで、成体ニューロンにおけるニューロンのエンドサイトーシス輸送を測定するために挑戦してきた。ここでは、急性の脳切片における表面タンパク質発現のex vivoでの急速な変化を測定するための非常に効果的な、定量的な方法を記載している。

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K ビュー.  University of Massachusetts Medical School. 神経細胞の膜輸送を動的に原形質膜タンパク質の可用性と大幅に影響を与える神経伝達を制御します。今日まで、成体ニューロンにおけるニューロンのエンドサイトーシス輸送を測定するために挑戦してきた。ここでは、急性の脳切片における表面タンパク質発現のex vivoでの急速な変化を測定するための非常に効果的な、定量的な方法を記載している。 

ビデオ: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

神経科学

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

GABA A受容体の膜局在化および輸送を研究するために、α-ブンガロトキシン結合部位タグを使用して

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

スライスこれホット：生理的温度における急性成人の脳スライス

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 タンパク質デコンボリューション顕微鏡を用いたマウス一次視床下部ニューロンにおける人身売買のライブ映像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivoショウジョウバエにおける内分泌シグナル伝達する脳の反応を可視化するためのカルシウム イメージング

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

げっ歯類の脳におけるタンパク質発現を測定するために近赤外蛍光と高解像度走査を用いること

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...