The authors are grateful to G. Barka (SunChrom GmbH, Friedrichsdorf, Germany) for his support and for providing the epoxy glue. Furthermore, the authors acknowledge Fresenius Medical Care (Bad Homburg, Germany) for supplying the Capillary Haemodiafilter FXCorDiax. Funding was obtained from Goethe University of Frankfurt.

1.準備PTFE、チューブ<ol><li> 2.5センチメートル片をPTFE-チューブを短くしてください。物理的寸法を推定するために、スケールの用紙を使用します。 （ 図3aを参照） </li><li>出口チューブの接続を容易にする角度で1エンディングをカットします。 （ 図3aを参照） </li><li>サンドペーパーの使用によるラフPTFE-チューブはエポキシ接着剤の付着を可能にします。 </li></ol>溶融シリカの準備2. <ol><li> 2.25センチメートル片に溶融シリカを短くしてください。 （ 図3aを参照） </li></ol> 3. PTFE-チューブ内にフューズドシリカを挿入<ol><li>平らな端1センチの距離でそれを貫通する中空のPTFE繊維に30 Gカニューレを挿入します。プロセスを簡略化するPTFE-チューブを曲げます。 （ 図3bを参照してください） </li><li>ガイダンスとして、カニューレを使用して、PTFE、チューブに溶融シリカを挿入します。 （ 図3cを参照してください） </li><li>ゆっくりカニューレを除去し、溶融シリカを固定します。 （参照してください。図3d） </li><li>溶融シリカは、平坦な端で5ミリメートル突出するようになっています。 （ 図3Eを参照してください） </li><li>シアノアクリレート接着剤により、穿刺部位での代わりに溶融シリカを接着して、一晩硬化することができます。 （ 図3Eを参照してください） </li></ol> 4.準備エポキシ接着剤（二成分接着剤） <ol><li>微細針の助けによってブラックエポキシ接着剤70部に黄色の硬化剤30部を加え、穏やかに混ぜます。これは、直接使用できるようになりました。 </li></ol> 5.膜を適用する（すべてのさらなる工程は、顕微鏡下で行われなければならないとスケール紙は外形寸法を推定するために使用されます） <ol><li> PTFE-チューブ内に膜を引っ張り、鋭いメスを用いて5.5ミリメートルに膜を調整し、その後、溶融シリカの上に慎重にポリスルホン膜を引き出します。 （ 図3Fを参照してください） </li><li> 2mmの交換長さを定義するために細かいペンで膜上のマーキングポイントを設定します。膜の端から。 </li><li>微細針の助けを借りて、膜の先端部を覆うようにエポキシ接着剤の少量を適用します。 （図3グラムを参照してください） </li><li>エポキシ接着剤で）ステップ5.3で使用したのと同じ微細針の助けにより、透析膜とPTFE繊維との間の接合部を閉鎖。 （ 図3グラムを参照してください） </li><li> 2mmの交換長さを確保するために、マーキングポイントまで膜に十分なエポキシ接着剤を追加します。それが一晩硬化することができます。交換長さの決意する前に（ 図3gを参照 ）、膜5.5 mmです。膜上にエポキシ接着剤を塗布した後、膜の唯一の2ミリメートルは、拡散のために利用可能です。 </li></ol> 6.金属スリーブを適用します<ol><li>曲げペンチの助けによって、1cmの金属スリーブ片に25 G針をカットします。 </li><li>溶融シリカの残りの部分の上に1cmの金属スリーブ（25 G）を引き出します。金属スリーブは、後MICRODIA入口とチューブの接続を可能にします溶解プローブ。 （ 図3Hを参照してください） </li><li>残りのエポキシ接着剤で金属スリーブとPTFE-チューブとの間の接合部をシールします。それは、少なくとも24時間硬化することができます。 （ 図3Iを参照してください） </li><li>追加の固定および安定化のためのプローブの分岐部の周りにホットグルーを適用します。 注：プローブの入口内部容積は0.045μLと出口の内部容積は2.5μlです。したがって、1μL/分の流速で、それは、透析液の収集を開始するために導入管を接続した後、約2.5分を要します。本プロトコルでは、プローブの組み立て手順は、95％信頼性の高い微小透析プローブをもたらします。 </li></ol> インビトロ回復実験の7.パフォーマンス<ol><li> 100を含有CSFに、30kDaのカットオフおよびaCSFのが別々の実験で1 mMグルコースおよび1 mMの乳酸を含むからなる原液に2mmの交換長さのプローブを浸しnmのATPおよび100 nMのアセチルコリン。 </li><li> aCSFの有するプローブを灌流。 1μL/分に流量を設定し、室温（22℃）で実験を行います。プローブは原液で1時間平衡化させ、その後、サンプルを30分毎に収集し、測定まで-80℃で保存してください。 </li><li> in vitroでの回収率の計算のために、原液の既知の検体濃度と透析液中の分析物の濃度を比較します。原液対透析液中の物質濃度の割合として結果を表現します。 </li></ol>

<ol>
	<li>Feldberg, W., Malcom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+site+of+action+of+tubocurarine+when+applied+via+the+cerebral+ventricles.">Experiments on the site of action of tubocurarine when applied via the cerebral ventricles.</a> J Physiol. 149, 58-77 (1959).</li><li>Gaddum, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Push-pull+cannulae.">Push-pull cannulae.</a> J Physiol. 155 (1 P), (1961).</li><li>Bito, L., Davson, H., Levin, E., Murray, M., Snider, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentrations+of+free+amino+acids+and+other+electrolytes+in+cerebrospinal+fluid,+in+vivo+dialysate+of+brain,+and+blood+plasma+of+the+dog.">The concentrations of free amino acids and other electrolytes in cerebrospinal fluid, in vivo dialysate of brain, and blood plasma of the dog.</a> J Neurochem. 13 (11), 1057-1067 (1966).</li><li>Delgado, J. M., Lerma, J., Mart&#237;n del R&#237;o, R., Sol&#237;s, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialytrode+technology+and+local+profiles+of+amino+acids+in+the+awake+cat+brain.">Dialytrode technology and local profiles of amino acids in the awake cat brain.</a> J Neurochem. 42 (5), 1218-1228 (1984).</li><li>Ungerstedt, U., Pycock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+correlates+of+dopamine+neurotransmission.">Functional correlates of dopamine neurotransmission.</a> Bull Schweiz Akad Med Wiss. 30 (1-3), 44-55 (1974).</li><li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis--principles+and+applications+for+studies+in+animals+and+man.">Microdialysis--principles and applications for studies in animals and man.</a> J Intern Med. 230 (4), 365-373 (1991).</li><li>Bourne, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+microdialys:+30+years+as+a+tool+for+the+neuroscientist.">Intracerebral microdialys: 30 years as a tool for the neuroscientist.</a> Clin Exp Pharmacol Physiol. 30 (1-2), 16-24 (2003).</li><li>Lietsche, J., Gorka, J., Hardt, S., Karas, M., Klein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-built+microdialysis+probes+with+improved+recoveries+of+ATP+and+neuropeptides.">Self-built microdialysis probes with improved recoveries of ATP and neuropeptides.</a> J Neurosci Methods. 237, 1-8 (2014).</li><li>Chefer, V. I., Thompson, A. C., Zapata, A., Shippenberg, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+Brain+Microdialysis.">Overview of Brain Microdialysis.</a> Curr Protoc Neurosci. 7, Unit 7.1 (2009).</li><li>Neurochem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Microdialysis.">Brain Microdialysis.</a> J. Neurochem. 52 (6), 1667-1679 (1989).</li><li>Jolly, D., Vezina, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+microdialysis+in+the+rat:+low+cost+and+low+labor+construction+of+a+small+diameter,+removable,+concentric-style+microdialysis+probe+system.">In vivo microdialysis in the rat: low cost and low labor construction of a small diameter, removable, concentric-style microdialysis probe system.</a> J Neurosci Methods. 68 (2), 259-267 (1996).</li><li>Sumbria, R., Klein, J., Bickel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+depression+of+energy+metabolism+after+microdialysis+probe+implantation+is+distinct+from+ischemia-induced+changes+in+mouse+brain.">Acute depression of energy metabolism after microdialysis probe implantation is distinct from ischemia-induced changes in mouse brain.</a> Neurochem Res. 36, 109-116 (2010).</li></ol>

The authors have nothing to disclose.

図3は、例示的なプローブアセンブリを示しています。正しい内側と外側の直径寸法は、密接にチューブのために観察されなければならない（OD 1.6ミリメートル、ID 350ミクロン）、溶融シリカ（OD 105ミクロン; ID 40ミクロ​​ン）と透析膜（OD 245ミクロン; ID 210ミクロン）。これは、膜および（105ミクロン）の溶融シリカとの間の空間を維持することも重要であると同様に（105ミク...

微小透析は、神経科学の研究で​​一般的に使用される技術です。過去50年の間に、最小限の侵襲微小透析技術は、継続的に細胞外空間に低分子量化合物の局所濃度を監視するための十分に確立された方法になるように改善されました。ほぼすべての間質組織液が自由に動く動物で調べることができます。 Gaddumは、1960年代にプッシュプル法を導入しました。彼はフェルトベルグらからのアプローチを変更しました。ツボクラリンは側脳室で終わるカニューレを通して灌流カニューレ1を介して、また流出物を収集しました。 Gaddumは、2つの同心のスチール針からなるカニューレは別個の脳領域に移植し、同時にチップ2の周囲のニューロンから放出神経伝達物質を除去しながら液で灌流されたプッシュプル法を開発しました。残念ながら、組織DAMA先端の周りにカニューレによって引き起こさGEは、この方法の適用を制限しました。この方法のさらなる前進として、尾藤と共同研究者は、収集した溶液を透析膜によって周囲の組織から分離された透析バッグ法を導入しました。彼らは犬の首の皮下組織に透析嚢を移植しました。透析袋の内容は、無タンパク質であり、イオンおよびアミノ酸のための多くの3週間後に分析することができました。次の開発は、もともと1972年4デルガドによって記述された原始的な微小透析プローブ、dialytrodeだった。それは小さく、あまり組織5を変位となるように最後に、Ungerstedtらは微小透析プローブの設計を改善しました。 同心の微小透析プローブは、毛細血管と同じように動作します。システムは常に関心のある検体を欠くながら周囲の組織液のイオン組成をフィーチャー液で灌流されます。目外液または組織にさらされる電子の透析膜は半透過性です。それは、その濃度勾配6に沿ってプローブ内の物質の受動拡散を可能にします。透過性は、そのような化合物の分子量、形状、電荷およびpHなどの多くの変数に依存しています。また、膜材料、膜および流量7の孔サイズの特性に限定されます。 図1及び図2は、同心円状微小透析プローブを示します。灌流液は、溶融シリカを囲む金属スリーブ、への入口管を介して入ります。金属スリーブの内部では、溶融シリカに沿って下方に流れ、その先端にそれを残します。透析膜とポリテトラフルオロエチレン（PTFE）（例えばテフロンなど）-tubingとの間の空間に、灌流液は、その後、上方へ流れます。ここでは、組織からの物質の拡散は、膜を包囲するが発生します。透析液は、PTFを介してプローブを残しますチューブをコンセントに接続されており、回収することができるE-チューブ。 微小透析法は、 インビボ法の他に比べていくつかの利点を有します。プローブは透析液には酵素または細胞を含まない結果に物理的障壁を構成しています。したがって、そこに分析の前に溶出液の精製の必要がなく、分析物のない酵素分解は起こりません。酸化劣化は、チューブ中の分析物の通過の間に発生することができ、これは、多くの場合、灌流液に酸化防止剤（ 例えばアスコルビン酸）を添加することによって防止することができます。代替的に、神経ペプチドの酸化的損傷は、例えば、効率透析液8を収集するために先端で出口チューブを交換することによって抑制されました。透析液は、分析方法のほぼすべての種類を直接調査することができ、複数の分析物を同時に収集することができます。このシステムは、覚醒動物において使用することができ、ほぼすべての脳領域であることができます検討しました。また、プローブを通る薬剤の注入は（retrodialysis）が可能です。しかし、微小透析技術の限界もあります。やや低時間分解能は、神経伝達物質の変化に関するリアルタイムの情報を提供していません。それは、侵襲的技術であるため、プローブの移植は、外科的外傷および麻酔を引き起こす、神経伝達物質の濃度に影響を与えることができ、このステップ7,9,10の間に必要とされます。 透析液中の化合物濃度は、細胞外液中の実際の化合物の濃度のわずかな量を含みます。細胞外液中の未知の化合物濃度を計算するため、相対的なインビトロ回復を計算しなければなりません。個々のすべてのプローブのためのin vitroでの回収率の相対的な、すべての化合物の決意は、in vivoでの実験を開始する前に必要です。この目的のために、プローブを含有する溶液に浸漬します灌流液に対し、目的の分析物は、目的の分析物を欠く同じソリューションです。透析液中の化合物濃度を決意した後、このデータは、周囲の流体中のそれらの濃度に言及する必要がある。いくつかの物質のインビトロ回復測定を同時に実施することができる9。 多くの神経科学の研究グループは、異なる脳領域の細胞外空間内の神経伝達物質および代謝物を調査するために微小透析技術を使用しています。したがって、商業プローブが神経科学の研究環境に大きな需要があります。市販されているプローブの大きな利点は、高い機能性、信頼性です。商業プローブの実験設定は十分に確立されており、多くのよく知られた神経伝達物質および代謝物のために検証されます。しかし、市販の微小透析装置は高価であり、目に、アプリケーション11が少ない柔軟性を有するのに対し任意のアプリケーションに適合させることができ、10ドル未満のために製造することができる、微小透析プローブアセンブリが​​詳細に示されている作業です。このカスタムメイドのプローブは、様々な脳領域8内の調査のために試験同心微小透析プローブです。 異なる分子量（100-1,600ダの範囲）で、異なる物理化学的特性を比較すると物質のin vitro でのプローブの回収では 、分子量と分子量のグルコース、乳酸およびアセチルコリンのインビトロ回復決意200Da未満、ATP約500ダおよび神経ペプチドのアンジオテンシンII、千ダ上記分子量のサブスタンスPおよびソマトスタチンが行われます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Epoxy glue</td>
			<td>SunChrom GmbH, Fiedrichsdorf, Germany</td>
			<td></td>
			<td>&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Fused silica ID 40 &#181;m OD 105 &#181;m</td>
			<td>Ziemer-Chromatographie, Mannheim, Germany</td>
			<td>Art. No: 6.040105</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone membrane (haemodialysis filter FX Cor Diax 600)</td>
			<td>Fresenius Medical Care AG &#38; Co. KGaA, Bad Homburg, Germany</td>
			<td>REF: F00001593</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanacrylate glue (Pattex&#174; superglue plastic)</td>
			<td>Henkel AG&#38;Co. KGaA, D&#252;sseldorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEFLON-tubing 1.6 x 0.35 mm</td>
			<td>SunChrom&#174; GmbH, Friedberg, Germany</td>
			<td>Art. No: 969-195.219</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (Feather&#174; Surgical Blade No. 10)</td>
			<td>pfm medical ag, cologne, Germany</td>
			<td>Art. No: 07310</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>MEIJI Techno&#160;</td>
			<td>EMZ-8TR</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 11&#34; (0.3 x 25 mm) cannula&#160; Sterican&#174; Z</td>
			<td>B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>REF: 9324500</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 11/2&#34; (0.5 x 40 mm) cannula 100 Sterican&#174;</td>
			<td>B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>REF: 9186166</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine pen (Stabilo point 88 fine 0.4)</td>
			<td>Schwan-STABILO Schwanh&#228;u&#223;er GmbH &#38; Co. KG</td>
			<td>Art. No: 88/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot glue&#160; (Glue sticks ULTRA Power x 11 mm)</td>
			<td>Steinel&#174; GmbH, Herzebrock-Clarholz, Germany</td>
			<td>Art. No: 4007841046910</td>
			<td></td>
		</tr>
		<tr>
			<td>Sandpaper P60 230 x 280</td>
			<td>Robert Bosch GmbH, Gerlingen-Schillerh&#246;he, Germany</td>
			<td>Catalog Number: 2608605397</td>
			<td></td>
		</tr>
	</tbody>
</table>

custom-made microdialysis probe design

微小透析は、神経科学の研究で​​一般的に使用される技術です。したがって、商業的プローブは、脳脊髄液中の、生理的薬理学的および病理学的変化を監視するための大きな需要があります。残念ながら、商業的プローブは、公的機関の研究グループのために高価です。本研究では、プローブアセンブリは、10ドル未満のために信頼性の高い、同心、カスタムメイドの微小透析プローブを構築するために詳細に説明します。微小透析プローブは、30kDaの分子カットオフを有するポリスルホン膜で構成されています。プローブは、in vitro（100-1,600ダの範囲内の）異なる分子量、異なる物理化学的特性を有する物質の回収率が比較されています 。プローブは、低分子化合物のグルコース、乳酸塩、アセチルコリンおよびATPのための約20％のin vitroでの回復をもたらす。2〜6％に1,000-1,600ダ量との間の分子量を有する神経ペプチドのためのin vitroでの回収率。したがって、より高い分子W一方神経ペプチドの8がインビトロ回復値が低下し、分子量の低い範囲（最大500ダ）中の化合物の透析は、in vitroでの回収率には大きな影響を与えません。本発明の方法は、他のカットオフ値と膜材料と透析膜の使用を可能にします。したがって、このカスタムメイドのプローブアセンブリは、広い分子量範囲の物質を透析するのに十分な柔軟性の利点を有します。ここでは、マウスとラットの脳領域における微小透析に適用可能である2mmの交換長と微小透析プローブをご紹介します。しかし、プローブの寸法は簡単に、より大きな動物で使用する大きな交換長さに適合させることができます。

同心のカスタムメイドの微小透析プローブは、30kDaの分子カットオフを有するポリスルホン膜で構成されています。プローブアセンブリは、 図3に示されています。 それは、それぞれ、19.10±1.2％および21.2±1.6％の小さなエネルギー代謝グルコース（180.16ダ）と乳酸（112.06ダ）のためのin vitroでの回復を示しました。ダ181.66の分子量を有する正に荷電し?...

Watch this Scientific Journal Video about Custom-made Microdialysis Probe Design at JoVE.com

Custom-made Microdialysis Probe Design

微小透析は、神経科学の研究で​​一般的に使用される技術です。したがって、商業的プローブは素晴らしいdemand.Inにプローブアセンブリは、10ドル未満のために信頼性の高い、同心、カスタムメイドの微小透析プローブを構築するために詳細に説明されているこの作品です。

カスタムメイドマイクロダイアリシスプローブの設計

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.9K ビュー.  Goethe University of Frankfurt. 微小透析は、神経科学の研究で​​一般的に使用される技術です。したがって、商業的プローブは素晴らしいdemand.Inにプローブアセンブリは、10ドル未満のために信頼性の高い、同心、カスタムメイドの微小透析プローブを構築するために詳細に説明されているこの作品です。 

ビデオ: Custom-made Microdialysis Probe Design

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

水迷路における同時神経刺激および記録のためのコスト効率のヘッドステージの設計と構築

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

神経科学

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

ガスクロマトグラフィーによるオペラントエタノール自己投与とエタノール判別中エタノールのマイクロダイアリシス

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海馬のインスリンおよびマイクロインジェクション<emインビボ&gt;</em空間記憶テスト中&gt;マイクロダイアリシス

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

電子プローブX線マイクロアナリシスによるニューロンにおける総カルシウムの測定

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

リアルタイム電気生理学：プローブ神経ダイナミクスとその先にクローズド·ループ·プロトコルの使用

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

生体内で高分子量カットオフと間質液を脳から大規模な細胞外タンパク質を収集するマイクロダイアリシス法プローブします。

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由行動ラットの脳波記録中に興奮性アミノ酸の透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

シンプルかつ強力な可視化と薬理学的ターゲットの評価法として放射線写真法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to altered neurotransmission. Indeed, cerebral microdialysis studies in rodents have demonstrated an excessive extracellular glutamate release in the hippocampus within the first 10 min following trauma. Microdialysis offers the clear advantage of in vivo neurotransmitter continuous sampling while not having to sacrifice the animal. In addition to the aforementioned technique, a closed head injury model that exerts rapid acceleration and deceleration of the head and torso is needed, as such a factor is not available in many other animal models. The Wayne State weight-drop model mimics this essential component of human craniocerebral trauma, allowing the induction of an impact on the head of an unrestrained rodent with a falling weight. Our novel and translational rat model combines cerebral microdialysis with the Wayne State weight-drop model to study, in lightly anesthetized and unrestrained adult rats, the acute changes in extracellular neurotransmitter levels following concussion. In this protocol, the microdialysis probe was inserted inside the hippocampus as region of interest, and was left inserted in the brain at impact. There is a high density of terminals and receptors in the hippocampus, making it a relevant region to document altered neurotransmission following concussion. When applied to adult Sprague-Dawley rats, our combined model induced increases in hippocampal extracellular glutamate concentrations within the first 10 min, consistent with the previously reported post-concussion symptomology. This combined weight-drop model provides a reliable tool for researchers to study early therapeutic responses to concussions in addition to repetitive brain injury, since this protocol induces a closed-head mild trauma.

Neurotransmitter alteration is a mechanism of neural dysfunction that occurs after concussion and contributes to the sometimes-catastrophic long-term consequences. This rat model combines microdialysis, allowing in vivo neurotransmitter quantification, with a weight-drop technique exerting rapid acceleration and deceleration of the head and torso, an important factor of human craniocerebral trauma.

生体内脳微小透析における力と回転を組み合わせた脳震盪の新規および翻訳ラットモデル

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to ...

Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, at a subliminal perceptual level, and related brain mechanisms can be experimentally isolated by presenting the stimuli quickly. Subliminal presentation of faces that belong to different ethnicity groups, races, or gender has been shown to be successful in investigating social implicit responses. However, many implicit responses are based on knowledge previously gained about the faces (e.g., sexual orientation, political views, and socioeconomic status) and not solely on physical appearance. Here, a novel method called post-movie subliminal measurement (PMSM) is presented. When watching a socially engaging movie, a spectator gains knowledge about the protagonist and becomes familiar with his/her identity and world views. When the face of the protagonist is presented subliminally after the movie, it evokes an implicit neural response depending on what is learned about the protagonist. With a vast number of movies available, each depicting a variety of people with different identities, the PMSM method enables investigation of the brain&#39;s complex implicit biases in a manner that resembles real-life social perceptions.

Post-Movie Subliminal Measurement PMSM, for Investigating Implicit Social Bias

This protocol describes the use of movies to investigate brain mechanisms underlying implicit social biases during functional magnetic resonance imaging. When the face of a protagonist is presented after a movie subliminally, it evokes an implicit response based on knowledge of the protagonist gained during the movie.

暗黙的な社会的バイアスを調査するためのポストムービーサブリミナル測定(PMSM)

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, ...