著者は、このプロジェクトに関する技術サポートとガイダンスを提供してくれたBryan Krauseに感謝します。
この研究は、国際麻酔研究学会(IMRAからAR)、国立衛生研究所(R01 GM109086からMIB)、およびウィスコンシン大学マディソン校の医学部および公衆衛生学部麻酔科の支援を受けました。

<ol>
	<li>Baumgart, J. P., 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=Isoflurane+inhibits+synaptic+vesicle+exocytosis+through+reduced+Ca2++influx,+not+Ca2+-exocytosis+coupling.">Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.</a> Proceedings of the National Academy of Sciences U.S.A. 112 (38), 11959-11964 (2015).</li><li>Herring, B. E., Xie, Z., Marks, J., Fox, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+inhibits+the+neurotransmitter+release+machinery.">Isoflurane inhibits the neurotransmitter release machinery.</a> Journal of Neurophysiology. 102 (2), 1265-1273 (2009).</li><li>Xie, Z., 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=Interaction+of+anesthetics+with+neurotransmitter+release+machinery+proteins.">Interaction of anesthetics with neurotransmitter release machinery proteins.</a> Journal of Neurophysiology. 109 (3), 758-767 (2013).</li><li>Crick, F., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+consciousness.">A framework for consciousness.</a> Nature Neurosciences. 6 (2), 119-126 (2003).</li><li>Koch, C., Massimini, M., Boly, M., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+consciousness:+progress+and+problems.">Neural correlates of consciousness: progress and problems.</a> Nature Reviews Neurosciences. 17 (5), 307-321 (2016).</li><li>Dehaene, S., Changeux, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+approaches+to+conscious+processing.">Experimental and theoretical approaches to conscious processing.</a> Neuron. 70 (2), 200-227 (2011).</li><li>Friston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+theory+of+cortical+responses.">A theory of cortical responses.</a> Philosophical Transactions of the Royal Society of London B Biological Sciences. 360 (1456), 815-836 (2005).</li><li>Mashour, G. A., Hudetz, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-Up+and+Top-Down+Mechanisms+of+General+Anesthetics+Modulate+Different+Dimensions+of+Consciousness.">Bottom-Up and Top-Down Mechanisms of General Anesthetics Modulate Different Dimensions of Consciousness.</a> Frontiers in Neural Circuits. 11, 44 (2017).</li><li>Voss, L. J., Garcia, P. S., Hentschke, H., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Effects+of+General+Anesthetics+on+Cortical+Network+Activity+Using+Ex+Vivo+Preparations.">Understanding the Effects of General Anesthetics on Cortical Network Activity Using Ex Vivo Preparations.</a> Anesthesiology. 130 (6), 1049-1063 (2019).</li><li>Redinbaugh, M. 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=Thalamus+Modulates+Consciousness+Via+Layer-Specific+Control+of+Cortex.">Thalamus Modulates Consciousness Via Layer-Specific Control of Cortex.</a> Neuron. 105 (4), 0896 (2020).</li><li>Carhart-Harris, R. L., Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REBUS+and+the+Anarchic+Brain:+Toward+a+Unified+Model+of+the+Brain+Action+of+Psychedelics.">REBUS and the Anarchic Brain: Toward a Unified Model of the Brain Action of Psychedelics.</a> Pharmacological Reviews. 71 (3), 316-344 (2019).</li><li>Mak-McCully, R. A., 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=Coordination+of+cortical+and+thalamic+activity+during+non-REM+sleep+in+humans.">Coordination of cortical and thalamic activity during non-REM sleep in humans.</a> Nature communications. 8 (1), 15499 (2017).</li><li>Alkire, M. T., Hudetz, A. G., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consciousness+and+anesthesia.">Consciousness and anesthesia.</a> Science. 322 (5903), 876-880 (2008).</li><li>Sanders, R. D., Maze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noradrenergic+trespass+in+anesthetic+and+sedative+states.">Noradrenergic trespass in anesthetic and sedative states.</a> Anesthesiology. 117 (5), 945-947 (2012).</li><li>Hemmings, H. C., 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=Towards+a+Comprehensive+Understanding+of+Anesthetic+Mechanisms+of+Action:+A+Decade+of+Discovery.">Towards a Comprehensive Understanding of Anesthetic Mechanisms of Action: A Decade of Discovery.</a> Trends in Pharmacological Sciences. 40 (7), 464-481 (2019).</li><li>Nourski, K. V., 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=Auditory+Predictive+Coding+across+Awareness+States+under+Anesthesia:+An+Intracranial+Electrophysiology+Study.">Auditory Predictive Coding across Awareness States under Anesthesia: An Intracranial Electrophysiology Study.</a> Journal of Neurosciences. 38 (39), 8441-8452 (2018).</li><li>Liu, X., 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=Propofol+disrupts+functional+interactions+between+sensory+and+high-order+processing+of+auditory+verbal+memory.">Propofol disrupts functional interactions between sensory and high-order processing of auditory verbal memory.</a> Human Brain Mapping. 33 (10), 2487-2498 (2012).</li><li>Hentschke, H., Raz, A., Krause, B. M., Murphy, C. A., Banks, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+cortical+network+activity+by+the+general+anesthetic+isoflurane.">Disruption of cortical network activity by the general anesthetic isoflurane.</a> British Journal of Anaesthesiology. 119 (4), 685-696 (2017).</li><li>Lee, U., 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=Disruption+of+frontal-parietal+communication+by+ketamine,+propofol,+and+sevoflurane.">Disruption of frontal-parietal communication by ketamine, propofol, and sevoflurane.</a> Anesthesiology. 118 (6), 1264-1275 (2013).</li><li>Ferrarelli, F., 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=Breakdown+in+cortical+effective+connectivity+during+midazolam-induced+loss+of+consciousness.">Breakdown in cortical effective connectivity during midazolam-induced loss of consciousness.</a> Proceedings of the National Academy of Science U.S.A. 107 (6), 2681-2686 (2010).</li><li>Ku, S. W., Lee, U., Noh, G. J., Jun, I. G., Mashour, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+inhibition+of+frontal-to-parietal+feedback+connectivity+is+a+neurophysiologic+correlate+of+general+anesthesia+in+surgical+patients.">Preferential inhibition of frontal-to-parietal feedback connectivity is a neurophysiologic correlate of general anesthesia in surgical patients.</a> PLoS.One. 6 (10), 25155 (2011).</li><li>Lee, 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=Network+Properties+in+Transitions+of+Consciousness+during+Propofol-induced+Sedation.">Network Properties in Transitions of Consciousness during Propofol-induced Sedation.</a> Scientific Reports. 7 (1), 16791 (2017).</li><li>Murphy, 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=Propofol+anesthesia+and+sleep:+a+high-density+EEG+study.">Propofol anesthesia and sleep: a high-density EEG study.</a> Sleep. 34 (3), 283-291 (2011).</li><li>Murphy, C., Krause, B., Banks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+effects+of+isoflurane+on+cortico-cortical+feedback+afferent+responses+in+murine+non-primary+neocortex.">Selective effects of isoflurane on cortico-cortical feedback afferent responses in murine non-primary neocortex.</a> British Journal of Anaesthesiology. 123 (4), 488-496 (2019).</li><li>Raz, A., 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=Preferential+effect+of+isoflurane+on+top-down+versus+bottom-up+pathways+in+sensory+cortex.">Preferential effect of isoflurane on top-down versus bottom-up pathways in sensory cortex.</a> Frontiers in System Neurosciences. 8, 191 (2014).</li><li>Schrouff, 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=Brain+functional+integration+decreases+during+propofol-induced+loss+of+consciousness.">Brain functional integration decreases during propofol-induced loss of consciousness.</a> Neuroimage. 57 (1), 198-205 (2011).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457 (7233), 1142-1145 (2009).</li><li>Cruikshank, S. J., Urabe, H., Nurmikko, A. V., Connors, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-Specific+Feedforward+Circuits+between+Thalamus+and+Neocortex+Revealed+by+Selective+Optical+Stimulation+of+Axons.">Pathway-Specific Feedforward Circuits between Thalamus and Neocortex Revealed by Selective Optical Stimulation of Axons.</a> Neuron. 65 (2), 230-245 (2010).</li><li>Gredell, J. A., Turnquist, P. A., MacIver, M. B., Pearce, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+diffusion+and+partition+coefficients+of+propofol+in+rat+brain+tissue:+implications+for+studies+of+drug+action+in+vitro.">Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro.</a> BJA: British Journal of Anaesthesia. 93 (6), 810-817 (2004).</li><li>Benkwitz, C., 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=Determination+of+the+EC50+amnesic+concentration+of+etomidate+and+its+diffusion+profile+in+brain+tissue:+implications+for+in+vitro+studies.">Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.</a> Anesthesiology. 106 (1), 114-123 (2007).</li><li>Franks, N. P., Lieb, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+actions+of+volatile+general+anaesthetics+at+molecular+and+cellular+levels.">Selective actions of volatile general anaesthetics at molecular and cellular levels.</a> British Journal of Anaesthesia. 71 (1), 65-76 (1993).</li><li>Honemann, C. W., Washington, J., Honemann, M. C., Nietgen, G. W., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partition+coefficients+of+volatile+anesthetics+in+aqueous+electrolyte+solutions+at+various+temperatures.">Partition coefficients of volatile anesthetics in aqueous electrolyte solutions at various temperatures.</a> Anesthesiology. 89 (4), 1032-1035 (1998).</li><li>Au - Segev, A., Au - Garcia-Oscos, F., Au - Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> Journal of Visualized Experiments. (112), e54024 (2016).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophysical Journal. 96 (5), 1803-1814 (2009).</li><li>Lin, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+channelrhodopsin+variants:+features,+limitations+and+future+developments.">A user's guide to channelrhodopsin variants: features, limitations and future developments.</a> Experimental Physiology. 96 (1), 19-25 (2011).</li><li>Banks, M. I., Pearce, R. 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著者は開示するものは何もありません。

この原稿では、ex vivo脳スライスにおいて選択的に活性化された求心性経路に対する細胞内および細胞外の応答を評価するためのプロトコルが記載されている。
光遺伝学的ツールと並列記録スキームの使用により、研究者は、遠隔脳領域からの求心性入力に対する局所集団の応答を調査し、同時に介在ニューロンの標的集団から記録することができます。光遺伝学的技術?...

揮発性麻酔薬は、1世紀以上にわたってさまざまな臨床および学術環境で遍在的に使用されてきました。異なるクラスの麻酔薬は、独特の、しばしば重複しない分子標的1,2,3を持っていますが、それらのほとんどすべてが無意識を生み出します。それらの行動効果は非常に予測可能ですが、麻酔薬が意識喪失を誘発するメカニズムはほとんど知られていません。麻酔薬は、皮質視床回路上の作用を介して意識のレベルと内容の両方に最終的に影響を及ぼし、皮質階層全体の情報の統合を混乱させる可能性があります4,5,6,7,8,9。より広義には、コルチコ視床回路の調節は、実験的に10または薬理学的に11の変化した意識状態において役割を果たす可能性があり、睡眠12および意識の病態生理学的障害13,14にも関与し得る。
麻酔中の意識の喪失と回復の根底にあるメカニズムのとらえどころのないことは、細胞、ネットワーク、およびシステムレベルでの麻酔薬の非線形の相乗作用に部分的に起因する可能性があります15。イソフルランは、例えば、選択された脳領域16、17、18内の活動を抑制し、遠隔脳領域19、20、21、22、23間の接続性を損ない、経路特異的な様式でシナプス応答を減少させる24、25。.分子レベルからシステムレベルまで、麻酔薬のどの効果が意識喪失をもたらすために必要または十分であるかは不明である。非侵襲的技術を用いた意識の実質的な臨床調査19,20,26に加えて、実験家が意識的経験に従属する明確な細胞およびネットワークの相互作用を解きほぐそうとすることが重要です。
無傷の脳に見られる複雑な相互作用を単純化することにより、ex vivo脳スライスは、脳の動的システムの孤立した構成要素の研究を可能にします9。縮小スライス調製は、局所神経回路の比較的無傷の解剖学的構造の利点とin vitro操作の多様性を兼ね備えています。しかし、最近まで、方法論的制約により、脳スライスにおける長距離入力のシナプスおよび回路特性の研究が妨げられていました27,28。コルチコ視床線維路の曲がりくねった経路は、電気刺激によって独立した求心性経路の活性化をほとんど不可能にしました。
脳スライス製剤に対する麻酔薬の効果を調査することは、さらなる課題を提示する。無傷の呼吸器系および循環器系がない場合、麻酔薬を入浴させ、濃度を推定効果部位の濃度に注意深く一致させる必要があります。.多くの静脈内麻酔薬にとって、組織内の平衡化速度が遅いため、従来の薬理学的調査は面倒です29,30。ex vivo製剤中の揮発性ガス麻酔薬の効果を調査することは、より扱いやすいですが、課題もあります。これらには、吸入分圧用量を水性濃度に変換すること、および人工脳脊髄液31を介した組織への薬物の修正送達システムの必要性が含まれる。
ここでは、研究者が揮発性麻酔薬イソフルランの十分に文書化された物理化学的特性を利用して、ex vivo脳スライスへの薬物送達を行い、高い時空間分解能で皮質領域への経路および層特異的入力を活性化し、同時に実施する方法について説明します 層流記録と標的パッチクランプ記録 ニューロンの選択された集団から。これらの手順を組み合わせることで、研究者は、シナプスレベルからローカルネットワークレベルまで、いくつかの観察可能な電気生理学的応答特性における揮発性麻酔薬誘発性の変化を測定できます。

<table>
	<tbody>
		<tr>
			<td>2.5x broadfield objective lens</td>
			<td>Olympus</td>
			<td>MPLFLN2.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>40x water immersion objective lens</td>
			<td>Olympus</td>
			<td>LUMPLFLN40XW</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 mixture</td>
			<td>Airgas</td>
			<td>Z02OX95R2003045</td>
			<td></td>
		</tr>
		<tr>
			<td>A16 probe</td>
			<td>NeuroNexus</td>
			<td>A16x1-2mm-100-177-A16</td>
			<td>16-channel probe</td>
		</tr>
		<tr>
			<td>AAV2-hSyn-hChR2(H134R)-EYFP</td>
			<td>Karl Deisseroth Lab, UNC Vector Core</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic gas monitor (POET II)</td>
			<td>Criticare</td>
			<td>602-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, Magnesium Salt</td>
			<td>Sigma Aldrich</td>
			<td>A9187</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>007914</td>
			<td>Cre-dependent tdTomato mouse</td>
		</tr>
		<tr>
			<td>B6;129P2-Pvalbtm1(cre)Arbr/J</td>
			<td>The Jackson Laboratory</td>
			<td>008069</td>
			<td>PV-Cre mouse</td>
		</tr>
		<tr>
			<td>Belly Dancer Shaker</td>
			<td>Thomas Scientific</td>
			<td>1210H86-TS</td>
			<td>for equilibration of sealed gas bags</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic brand</td>
			<td></td>
			<td>for silver chloriding patch clamp electrode</td>
		</tr>
		<tr>
			<td>Bupivicaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Dot Scientific</td>
			<td>DSC20010</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Capillary glass (patch clamp recordings)</td>
			<td>King Precision Glass, Inc.</td>
			<td>KG-33</td>
			<td>Borosilicate, ID: 1.1mm, OD: 1.7mm, Length: 90.0mm</td>
		</tr>
		<tr>
			<td>Capillary glass (viral injections)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>3.5&#34;</td>
		</tr>
		<tr>
			<td>Control of junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>SM8</td>
			<td>for control of junior micromanipulator</td>
		</tr>
		<tr>
			<td>Control of manipulators and shifting table</td>
			<td>Luigs and Neumann</td>
			<td>SM7</td>
			<td>for control of multichannel electrode and shifting table</td>
		</tr>
		<tr>
			<td>Digidata 1440A + Clampex 10</td>
			<td>Molecular Devices</td>
			<td>1440A</td>
			<td>Digitizer and software</td>
		</tr>
		<tr>
			<td>E-3603 tubing</td>
			<td>Fisher Scientific</td>
			<td>14171208</td>
			<td>for delivery of 95% O2/5% CO2 gas mixture to incubation chamber + application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Dot Scientific</td>
			<td>DSE57060</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>ERP-27 EEG Reference/Patch Panel</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Filling needle</td>
			<td>World Precision Instruments</td>
			<td>50821912</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Filter cube for imaging EYFP</td>
			<td>Olympus</td>
			<td>U-MRFPHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific</td>
			<td>09801E</td>
			<td>lay over slice template during preparation of tissue block</td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>2.5x2.5 Box filament</td>
		</tr>
		<tr>
			<td>Gas dispersion tube</td>
			<td>Sigma Aldrich</td>
			<td>CLS3953312C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe (100 mL)</td>
			<td>Sigma Aldrich</td>
			<td>Z314390</td>
			<td>for filling gas-sealed bags</td>
		</tr>
		<tr>
			<td>Gluconic Acid, Potassium Salt (K-gluconate)</td>
			<td>Dot Scientific</td>
			<td>DSG37020</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Dot Scientific</td>
			<td>DSG32040</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>GTP, Sodium Salt</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Headstage-probe adaptor</td>
			<td>NeuroNexus</td>
			<td>A16-OM16</td>
			<td>adaptor to connect 16-channel probe to headstage input</td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>VWR International</td>
			<td>76192-096</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Dot Scientific</td>
			<td>DSH75030</td>
			<td>ACSF,intracellular solution</td>
		</tr>
		<tr>
			<td>HS-16 Headstage</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (70%)</td>
			<td>VWR International</td>
			<td>101223-746</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>210-100 000 0090-R</td>
			<td>for manipulation of patch clamp electrode</td>
		</tr>
		<tr>
			<td>LED Light Source Control Module</td>
			<td>Mightex</td>
			<td>BLS-PL02_US</td>
			<td>optogenetic light source control</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Amplifier</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Lynx-8 Power Supply</td>
			<td>Neuralynx</td>
			<td>Retired</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4)</td>
			<td>Dot Scientific</td>
			<td>DSM24300</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>mCherry, Texas Red filter cube</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>for imaging tdTomato fluorescent reporter</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Fisher Scientific</td>
			<td>NC9044962</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Generic brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700A</td>
			<td>Molecular Devices/Axon Instruments</td>
			<td>700A</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Nitrogen (for air table)</td>
			<td>Airgas</td>
			<td>NI200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>501460083</td>
			<td>stretched over horseshoe of flattened platinum wire, slice rest on top of this during recordings</td>
		</tr>
		<tr>
			<td>Nylon, cut from pantyhose</td>
			<td>Generic brand</td>
			<td></td>
			<td>small piece to create slice platform in incubation chamber, single fibers to create platinum harp</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher Scientific</td>
			<td>NC1697520</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Dot Scientific</td>
			<td>307</td>
			<td>For transferring tissue to rig</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>VWR International</td>
			<td>BT124000</td>
			<td>2 cm, flattened, to make platinum harp</td>
		</tr>
		<tr>
			<td>Polygon400</td>
			<td>Mightex</td>
			<td>DSI-E-0470-0617-000</td>
			<td>optogenetic light delivery system, comes with PolyScan2 software</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Dot Scientific</td>
			<td>DSP41000</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Potassium Phosphate (KH2PO4)</td>
			<td>Dot Scientific</td>
			<td>DSP41200</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade (for vibratome)</td>
			<td>VWR International</td>
			<td>100492-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-361445</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealed gas bag</td>
			<td>Fisher Scientific</td>
			<td>109236</td>
			<td></td>
		</tr>
		<tr>
			<td>Shifting table for microscope</td>
			<td>Luigs and Neumann</td>
			<td>380FMU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (HCO3-)</td>
			<td>Dot Scientific</td>
			<td>DSS22060</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Dot Scientific</td>
			<td>DSS23020</td>
			<td>ACSF, intracellular solution</td>
		</tr>
		<tr>
			<td>Ssttm2.1(cre)Zjh/J (SOM-IRES-Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>013044</td>
			<td>SOM-Cre mouse</td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Kopf</td>
			<td>Model 902</td>
			<td>Dual Small Animal</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Staples</td>
			<td>886833</td>
			<td>to fix tissue block to specimen stage during slice preparation</td>
		</tr>
		<tr>
			<td>Surgical drill</td>
			<td>RAM Products Inc.</td>
			<td>DIGITALMICROTORQUE</td>
			<td>Microtorque II</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with LuerLock tip</td>
			<td>Fisher Scientific</td>
			<td>309628</td>
			<td>for application of pressure during patch clamping</td>
		</tr>
		<tr>
			<td>Syringe (1 mL) with slip tip</td>
			<td>WW Grainger, Inc.</td>
			<td>19G384</td>
			<td>for filling patch clamp pipettes</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>VWR International</td>
			<td>66064-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wypall towels</td>
			<td>Fisher Scientific</td>
			<td>19-042-427</td>
			<td></td>
		</tr>
	</tbody>
</table>

optogenetic activation of afferent pathways in brain slices and modulation of responses by volatile anesthetics

麻酔薬は、視床皮質回路に対する作用を介して意識に部分的に影響を及ぼします。しかし、揮発性麻酔薬がこれらの回路の明確な細胞およびネットワークコンポーネントにどの程度影響を与えるかは不明のままです。生体外脳スライスは、研究者が複雑なネットワークの個別のコンポーネントを調査し、誘発された反応に対する揮発性麻酔薬の影響の根底にある潜在的なメカニズムを解きほぐす手段を提供します。脳スライス中の潜在的な細胞タイプおよび経路特異的薬物効果を分離するには、研究者は求心性線維経路を独立して活性化し、重複しない細胞集団を特定し、揮発性麻酔薬を水溶液中の組織に適用できなければなりません。このプロトコルでは、ex vivo脳スライスにおける新皮質への2つの独立した求心性経路に対する光遺伝学的に誘発された応答を測定する方法が記載されている。細胞外応答はアッセイネットワーク活性に対して記録され、標的全細胞パッチクランプ記録はソマトスタチンおよびパルブアルブミン陽性介在ニューロンで行われます。細胞およびネットワーク応答を調節するための人工脳脊髄液を介した生理学的に関連する濃度のイソフルランの送達が記載されている。

プロトコルで説明されている手順のタイムラインを 図 1 に示します。高次皮質領域または非一次視床核から到着する皮質入力は、非一次視覚野24の層1で部分的に重複する終末野を有する。独立した視床皮質または皮質皮質求心性経路を単離するために、ChR2およびeYFP蛍光レポーターを含むウイルスベクターをPoまたはCgのいずれかに注入した。注射半径...

Watch this Scientific Journal Video about Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics at JoVE.com

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

Ex vivo脳スライスは、求心性入力に対する誘発反応に対する揮発性麻酔薬の効果を研究するために使用できます。光遺伝学は、視床皮質および皮質皮質求心性神経を非原発性新皮質に独立して活性化するために使用され、シナプスおよびネットワーク応答はイソフルランで調節されます。

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 ...

optogenetic-activation-afferent-pathways-brain-slices-modulation

Research

JoVE Journal

Neuroscience

2.1K ビュー.  University of Wisconsin. Ex vivo脳スライスは、求心性入力に対する誘発反応に対する揮発性麻酔薬の効果を研究するために使用できます。光遺伝学は、視床皮質および皮質皮質求心性神経を非原発性新皮質に独立して活性化するために使用され、シナプスおよびネットワーク応答はイソフルランで調節されます。

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

視床下部マウス脳スライスのGFPタグニューロンにおけるイメージングカルシウム応答

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

神経科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

CHEF-tdTomatoを使用してゼブラフィッシュ体性感覚ニューロンのOptogeneticアクティベーション

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

齧歯類の中枢神経系の生体光遺伝学的刺激

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

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 ...

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 ...

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

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

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

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

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

記録と電極アレイに結合された齧歯動物の脳スライスにおけるてんかん活動の変調

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

気相で、嗅覚による嗅覚受容体活性化の In Vitro モニタリング リアルタイム

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.