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 style="border-collapse:collapse;width:665pt" width="887">
	<colgroup>
		<col style="width:161pt" width="214" />
		<col style="width:140pt" width="187" />
		<col style="width:155pt" width="206" />
		<col style="width:210pt" width="280" />
	</colgroup>
	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Glass Borosilicate micropipettes</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">BF150-86-10</td>
			<td class="xl18" style="width:210pt" width="280">1.50 mm outer diameter; 0.86 mm inner diameter</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micropipette Puller</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MODEL P-97&#160;</td>
			<td class="xl18" style="width:210pt" width="280">Flaming/Brow Micropipette Puller</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micromanipulators</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MP-285</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Computer controlled Amplifier&#160;&#160;&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Multiclamp 700B</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Digital Acquisition system&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Digidata 1440A</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Imaging Camera</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl18" style="width:155pt" width="206">2115001</td>
			<td class="xl18" style="width:210pt" width="280">Inspection equipment</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Microscopy&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl20" style="width:155pt" width="206">Eclipse FN1&#160;</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Master 8</td>
			<td class="xl20" style="width:140pt" width="187">A.M.P.I.</td>
			<td class="xl20" style="width:155pt" width="206">Master-8 Pulse stimulator</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Vibratome Slicer</td>
			<td class="xl20" style="width:140pt" width="187">Leica</td>
			<td class="xl20" style="width:155pt" width="206">VT 1000S</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Picospritzer &#8546;</td>
			<td class="xl20" style="width:140pt" width="187">Parker Hannifin</td>
			<td class="xl20" style="width:155pt" width="206"></td>
			<td class="xl18" style="width:210pt" width="280">Pressure Systems for Ejection of 
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		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Razor blade</td>
			<td class="xl20" style="width:140pt" width="187">Gillette</td>
			<td class="xl20" style="width:155pt" width="206">74-S</td>
			<td class="xl20" style="width:210pt" width="280">FLYING EAGLE</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video monitor</td>
			<td class="xl20" style="width:140pt" width="187">Panasonic</td>
			<td class="xl20" style="width:155pt" width="206">WV-BM 1410</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">502 Glue</td>
			<td class="xl20" style="width:140pt" width="187">Deli</td>
			<td class="xl18" style="width:155pt" width="206">7146</td>
			<td class="xl20" style="width:210pt" width="280">Cyanoacrylate Glue</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Peristaltic pump</td>
			<td class="xl20" style="width:140pt" width="187">Shanghai JIA PENG Corporation</td>
			<td class="xl20" style="width:155pt" width="206">BT100-1F</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video Camera</td>
			<td class="xl20" style="width:140pt" width="187">Olympus America Medical</td>
			<td class="xl20" style="width:155pt" width="206">OLY-150</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">Transfer Pipets</td>
			<td class="xl18" style="width:140pt" width="187">Biologix</td>
			<td class="xl18" style="width:155pt" width="206">30-0138A1</td>
			<td class="xl20" style="width:210pt" width="280"></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

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

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

マウスの脳スライスにおけるニコチン性アセチルコリン受容体機能を研究するための薬物のローカルアプリケーション

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

器官型海馬スライスにおけるペア全細胞記録

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

脳スライスにおけるニューロン研究のための自動画像誘導パッチクランプの応用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

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

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

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

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

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

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 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