Name: whole-cell currents induced by puff application of gaba in brain slices
Uploaded: 2017-10-12T14:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

著者は以下の組織を感謝したい: 国家自然科学基金、中国の (31171018、31171355)、科学技術広東部 (2013KJCX0054)、広東省の自然科学基礎 (2014A030313418、2014A030313440)、広州科学と技術局 (201607010320)。

動物飼育および南中国師範大学、国立研究所によって確立された動物のケアのためのガイドラインによると動物の研究のための倫理委員会によって承認された手続きに従って実験を行ったすべての動物健康.
 1 です。 ソリューションの準備 <ol><li>表 1 に記載されている当社の出版された仕事の 7 で説明したコンポーネントを含む人工の標準の脳脊髄液 (アプライド) の準備 500 mL。
	<ol><li>使用きれいなガラス ビーカーを脱イオン水新鮮なアプライド溶液を調製します。脱イオン水でへらをきれいにし、NaCl、KCl、NaH を追加する使用する前に乾燥 2 PO 4 NaHCO 3 MgSO 4、D-グルコース、95% O 2/5% ソリューションを持つバブル後 CaCl 2 を追加、CO 2 脱イオン水 500 ml (としての詳細な表 1) 〜 20 分のための混合物。完全に溶解するまで攪拌します</li>。
		任意の沈殿せずオフに <li>、アプライドは完全にことを確認します。7.2 7.4 に pH を調整します。95% O 2 と 5% CO 2 〜 20 分のための混合ガス気泡</li>
	</ol></li> <li>スライス切断溶液について、表 2 の化学物質 7 250 mL を準備します。
	<ol><li>KCl、MgCl 2 NaH 2 PO 4 NaHCO 3 choline·Cl、D-グルコースを追加し、最後 250 mL の脱イオン水を 1.1.1 に示すように CaCl 2 を追加、1.1.1、1.1.2 で説明されているように溶解します</li>。
		<li>95% O 2/5% をバブル後の CO 2、冷たいと凍結切削ソリューションになるように 15 ~ 20 分冷凍庫にソリューションを配置します 。
		注: 我々 は 1-2 か月からのスライスを作るときこの切削ソリューションを使用-古いマウス;2 か月より古いマウスの式が異なる場合があります</li>。
		<li>エンドトキシン等張生理食塩水に溶解することにより準備組合ソリューション (0.9 %nacl)。LPS は腹腔内投与 (0.5 mg/kg)、2 h を後で記録を開始します</li>。
	</ol></li></ol> 2。前頭野スライスの準備 <ol><li>冷凍庫から切削ソリューションを取るし、スラッシュを取得するホモジナイズしてください。氷上ソリューションを配置し、常にバブルの 95% O 2/5% CO 2.</li>
	<li>大規模な鋭いはさみを使用して、すばやく説明 9 としてマウスの首をはねるし、すぐに氷が入ったビーカーに頭を入れて (0-4 ° C) 切削ソリューションです</li>。
	<li>高級はさみで鼻尾側方向に正中線に沿って頭蓋骨の上部を切り取り、外側頭蓋部分を削除します。冷たい切削ソリューションに高級ヘラとドロップで脳を削除します 。
	メモ: 手順 2.2 2.3 急速に実行する必要があります (理想的には &#60; 1 分) です</li>。
	<li>は、氷を入れた 9 cm シャーレの蓋の脳を配置します。冷たい切削ソリューションで脳をすすいでください。小脳およびかみそりの刃と嗅球切断します</li>。
	<li>試料へ適用シアノアクリ レート系接着剤、vibratome のホルダーです。慎重に、吻側側の接着剤のドロップに脳ブロックを配置し、すぐに冷たい切削溶液に浸します</li>。
	<li>水平平面と 15 度の角度に vibratome のブレード ホルダーを調整し、コロナ脳スライス 350 μ m を準備 (翼振動周波数 = 85 Hz、速度 0.1 mm/s を =).</li>
	<li>定数バブルで 95% O 2/5% (RT) で 1 時間インキュベート アプライド、満ちている回復室にスライス転送ピペットを使用して、CO 2.</li>
</ol> 3。全細胞パッチ記録 <ol><li>作るガラス ホウケイ酸用マイクロ ピペットについて電極として使用または引き手操作マニュアルの特定のガイドラインに従ってマイクロ ピペットをパフします。電極、チップ抵抗は 4-6 M の範囲の &#8486;、パフ用マイクロ ピペットは、2-5 μ m 範囲の先端の直径を持っている</li>。
	<li>は、ブロック高速 Na + チャネルおよびこうして活動電位、アプライドに + Na チャンネル遮断薬 (TTX、1 μ M) を追加し、商工会議所に蠕動ポンプによって録音室で 2-3 mL/分のこの溶液の一定流量を維持それを吸引。バブルのアプライド 95% O 2/5% CO 2 スライスの存続させるため常にします</li>。
	<li>は、スライスのサイズに合わせてカットされた微細先端変更したパスツール ピペットを使用して録音室に脳スライスを転送します。間隔の並列ナイロン スレッドでプラチナ スライス アンカーでスライスをカバー &#8805; プラットフォーム上のスライスを保持するために 2 mm を離れてします</li>。
	<li>記録マイクロ ピペットを詰めて内部ソリューション 7 (表 3)、10 の μ M、50 μ M、100 μ M (アプライドで解散) や車両制御としてアプライドで GABA とパフ マイクロ ピペットを埋める</li>。
	<li>の破片との閉塞を防ぐために、アプライドに記録電極を浸す前に 1 mL の注射器を使用して光の肯定的な圧力を適用されます。顕微鏡 (4 x 対物レンズ)、マイクロマニピュレーターを使用してヒント ( 図 1 A) のモニターで、ビデオ画像の中央に表示されるように記録電極とパフのマイクロ ピペットを置きます</li>。
	<li>はパフ マイクロ ピペットを徐々 に削減しながら顕微鏡焦点 (40 x 対物レンズ) を調整し、45 ° の角度で記録ニューロン上に配置します。パフ マイクロ ピペットの先端と 20-40 μ m ( 図 1 B) 間に記録されたニューロンとの間の距離を保ちます。ゆっくりと慎重に記録電極を神経細胞にアプローチし、肯定的な圧力を離します。ギガ オーム シールを作成する電極ホルダーに接続されているチューブを弱いと簡単な吸引を適用します</li>。
	<li>維持 0 時電圧 mV。ギガ オーム シールの形成後、高速を補うため、手動でまたは自動的に容量を低下します。全体セル モードに入る上記チューブを介して簡潔で強力な吸引を適用します</li>。
	<li>電圧クランプのレコード eIPSC モード。マスター 8 ステップ電圧によって制御される Picospritzer を使用してシングルまたはペアの GABA のパフ ( 図 2) を実現するパフ ピペットに低ガス圧 (窒素、4-6 psi) を適用します</li>。
	<li>ベスト eIPSC 結果 ( 図 3) を取得し、LPS 誘導性うつ病モデル ( 図 4) で eIPSC を測定パフ期間を変更します</li>。
</ol>

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li><li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain.">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li><li>Eyo, U. B., 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=Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus.">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li><li>Dickinson, G. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs.">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li><li>Lee, J., 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=Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons.">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33 (2012).</li><li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus.">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, 1163-1178 (2007).</li><li>Chen, M., 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=APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition.">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li><li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li><li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1.">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li><li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity.">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li><li>Huo, Q., 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=Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice.">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li><li>Zhou, W. L., Gao, X. B., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms.">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li></ol>

著者が明らかに何もありません。

パフ アプリケーション シナプス後受容体機能3,4、7の評価に広く使用、各実験では正確なコントロールが必要です。GABA パフ前頭皮質脳スライスの Ips (すなわち、eIPSCs) の誘導を示す細胞パッチクを含む手順をご紹介します。記録電極の抵抗が約 5 MΩ、パフ用マイクロ ピペットの先端の直径は 2-5 μ m パフ圧力 4 6 psi 間は: ?...

パッチ ・ クランプの技術、神経細胞の電気信号を調査するための主要なツールは、1970 年代の1,2で開発されました。この手法の主な利点は、どのように特定の治療法の知識を提供すること (例えば、薬理学的) 神経機能やリアルタイムに3チャンネルに変更可能性があります。全細胞脳スライス内の記録の中に神経細胞の機能薬理学的評価には、記録されているニューロンにアゴニストまたは特定の受容体の拮抗薬などの薬物のアプリケーションが必要です。このメソッドは、次のニューロン4の生理学的および病理学的特性のよりよい理解につながる特定の薬剤のアプリケーションに発生する神経の変化の識別を許します。薬理学的管理は、5またはパフ6を灌流といずれかを介して行うことができるが、後者より優れたテクニックです。特に: アプリケーションを急速にパフ (i) 増加薬濃度レベルに記録されたニューロンの膜受容体の脱感作を防止; その(ii) パフ薬物の量は非常に低く、したがって脳のスライスに投与された化学物質の任意の望ましくない影響を軽減入浴ソリューションで少し効果があるよう(iii) パフのプロトコルは、設定、保存、作る実験を非常に正確に再現。(パフ iv) アプリケーションは、アゴニスト/拮抗薬、特に、このような試薬が高価なまたは入手困難の経済的な使用を表します。
ここでは、鋭く準備脳スライス、比較的保存状態の良い脳の回路の利点は、準備で GABA を吹き込むことによって引き起こされる細胞流れを記録に焦点を当てます。パフ誘発抑制電流7を行う方法は、この資料に記載されます。セシウム (Cs+) を使用して-0 でニューロンを押しベースの内部ソリューション、mV、GABA パフ誘発を紹介する適切な技術的な詳細と抑制性シナプス電流 (eIPSCs)。リポ多糖 (LPS) 注入8、我々 は、eIPSC を示す誘発性うつ病のマウスモデルを用いた GABA パフにより誘発される振幅が大幅に車両のコントロールと比較して組合を注入したマウスのスライスで減少します。我々 の意図は、この資料ではパフ手法は脳スライスにおける神経活動の化学物質や化合物の薬剤効果の評価を目的とした研究に広く適用できる方法を表示する必要がありますです。

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

whole-cell currents induced by puff application of gaba in brain slices

薬理学的管理は、全細胞パッチク脳スライス内の記録を行う場合に通常使用されます。電気生理学的記録中に新薬承認申請の最善の方法の 1 つは、パフの技術は、薬理学的試薬の脳スライスにおける神経活動に及ぼす影響を研究するためです。パフ アプリケーションの最大の利点はこのように膜受容体の脱感作を防止記録サイト中薬物濃度が急速に増大します。パフのアプリケーションの使用の成功は、次の要素に注意: 薬の濃度、パフ マイクロ ピペット、パフ ピペットと記録、ニューロンの先端と期間の間の距離のパラメーターとパフを運転圧力 (1 平方インチあたりポンド psi)。この資料では、γ-アミノ酪酸 (GABA) 前頭皮質スライスのニューロンに息を切らしによって引き起こされる全細胞の流れを記録するためのステップバイ ステップの手順を説明します。特に、海馬、線条体など他の頭脳区域、細胞培養などの異なる製剤にもマイナー変更と同じ手順を適用できます。

パフ テクニックは、薬の特定の神経細胞の表面に特定の受容体に及ぼす影響の研究に研究できます。この記事では、GABA 受容体を介した電流に着目します。パフと録音用のマイクロ ピペットを図 1に示します。パフは、アプライドによって生成された物理的な圧力の記録されたニューロンに影響を排除するために、ショ糖パフ (?...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

脳スライスにおける GABA のパフ アプリケーションによって引き起こされる全細胞の流れ

薬理学的試薬が全細胞パッチ ・ クランプ記録中に投与することができ、その成功に不可欠な機能のさまざまな側面を強調、パフの手法について述べる。

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.3K ビュー.  South China Normal University. 薬理学的試薬が全細胞パッチ ・ クランプ記録中に投与することができ、その成功に不可欠な機能のさまざまな側面を強調、パフの手法について述べる。

ビデオ: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

神経科学

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

脳スライスにおける全細胞パッチクランプ記録

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脳スライスにおける神経樹状突起における 2 光子カルシウム イメージング

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Γ-アミノ酪酸受容体をモジュレート新規化合物の探索の方法を入力、神経伝達

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

齧歯動物および人間のティッシュの急性発作活動の生成とオン ・ デマンド開始

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脳スライスにおける求心性経路の光遺伝学的活性化と揮発性麻酔薬による応答の調節

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

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

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

マウス脳の水平海馬スライス

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

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

マウス脳スライスにおけるGABAパフによる全細胞電流の記録

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

	...

脳スライスにおける GABA のパフアプリケーションによって引き起こされる全細胞の流れ

要約

さらに動画を探す

この動画の章

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

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

脳スライスにおける全細胞パッチクランプ記録

脳スライスにおける神経樹状突起における 2 光子カルシウムイメージング

Γ-アミノ酪酸受容体をモジュレート新規化合物の探索の方法を入力、神経伝達

齧歯動物および人間のティッシュの急性発作活動の生成とオン・デマンド開始

脳スライスにおける求心性経路の光遺伝学的活性化と揮発性麻酔薬による応答の調節

マウス脳の水平海馬スライス

マウス脳スライスにおけるGABAパフによる全細胞電流の記録

脳スライスにおける GABA のパフ アプリケーションによって引き起こされる全細胞の流れ

要約

さらに動画を探す

この動画の章

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

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

脳スライスにおける全細胞パッチクランプ記録

脳スライスにおける神経樹状突起における 2 光子カルシウム イメージング

Γ-アミノ酪酸受容体をモジュレート新規化合物の探索の方法を入力、神経伝達

齧歯動物および人間のティッシュの急性発作活動の生成とオン ・ デマンド開始

脳スライスにおける求心性経路の光遺伝学的活性化と揮発性麻酔薬による応答の調節

マウス脳の水平海馬スライス

マウス脳スライスにおけるGABAパフによる全細胞電流の記録

脳スライスにおける GABA のパフアプリケーションによって引き起こされる全細胞の流れ

脳スライスにおける神経樹状突起における 2 光子カルシウムイメージング

齧歯動物および人間のティッシュの急性発作活動の生成とオン・デマンド開始