FONCICYT 000000000091984; Jeansson、セーデルストレームKönigska sjukhemmet、マーサLundqvistさんとシグルドOCHエルザの基礎この作品は、スウェーデンの医学研究評議会（K2009 - 75SX - 21028〜01 - 3、K2008 - 61X - 20700〜01 - 3）によって賄われていたGoljesミンヌ、および医学SLS - 95151のスウェーデンの社会。教授ジーンD.ブロック、UCLAは、感謝して原稿上で貴重なコメントで知られています。我々はビデオ顕微鏡を提供するための博士マイケルAndäng博士とヘレナJohard HCNチャネルブロッカーのため、教授とアブデルエルManiraに感謝する。 倫理的配慮： 動物のすべての実験はカロリンスカ研究所と&quot;ストックホルムのノーラDjurförsöksetiska Nämnd&quot;で定められたガイドラインおよび規制に準拠して行った。全ての動物実験は、動物へのあらゆる可能なストレスや不快感を最小限に抑える目的で行われます。

<ol>
	<li>Welsh, D. K., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suprachiasmatic+nucleus:+cell+autonomy+and+network+properties.">Suprachiasmatic nucleus: cell autonomy and network properties.</a> Annu Rev Physiol. 72, 551-577 (2010).</li><li>Green, D. J., Gillette, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+firing+rate+recorded+from+single+cells+in+the+rat+suprachiasmatic+brain+slice.">Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice.</a> Brain Res. 245, 198-200 (1982).</li><li>Shibata, S., Oomura, Y., Kita, H., Hattori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythmic+changes+of+neuronal+activity+in+the+suprachiasmatic+nucleus+of+the+rat+hypothalamic+slice.">Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice.</a> Brain Res. 247, 154-158 (1982).</li><li>Groos, G., Hendriks, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+electrical+discharge+of+rat+suprachiasmatic+neurones+recorded+in+vitro.">Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro.</a> Neurosci Lett. 34, 283-288 (1982).</li><li>Moore, R. Y., Klein, D., Moore, R., Reppert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+suprachiasmatic+nucleus+and+the+circadian+timing+system.">The suprachiasmatic nucleus and the circadian timing system.</a> Suprachiasmatic nucleus--The mind's clock. , 13-15 (1991).</li><li>Pol, A. N. V. a. n. d. e. 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+hypothalamic+suprachiasmatic+nucleus+of+rat:+intrinsic+anatomy.">The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy.</a> J Comp Neurol. 191, 661-702 (1980).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block, G. D., Sakaki, Y., Menaker, M., Tei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+central+and+peripheral+circadian+oscillators+in+transgenic+rats.">Resetting central and peripheral circadian oscillators in transgenic rats.</a> Science. 288, 682-685 (2000).</li><li>Geusz, M. E., Fletcher, C., Block, G. D., Straume, M., Copeland, N. G., Jenkins, N. A., Kay, S. A., RN, D. a. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+monitoring+of+circadian+rhythms+in+c-fos+gene+expression+from+suprachiasmatic+nucleus+cultures.">Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures.</a> Curr Biol. 7, 758-766 (1997).</li><li>Ko, C. H., Buhr, E. D., Siepka, S. M., Hong, H. K., Oh, W. J., Yoo, O. J., Menaker, M., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci U S A. 101, 5339-5346 (2004).</li><li>Yamazaki, S., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+luminescence+reporting+of+circadian+gene+expression+in+mammals.">Real-time luminescence reporting of circadian gene expression in mammals.</a> Methods Enzymol. 393, 288-301 (2005).</li><li>Yoshikawa, T., Yamazaki, S., Menaker, M. <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+preparation+time+on+phase+of+cultured+tissues+reveal+complexity+of+circadian+organization.">Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization.</a> J Biol Rhythms. 20, 500-512 (2005).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (1997).</li><li>Paxinos, G., Watson, 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> The rat brain in stereotaxic coordinates. , (1998).</li><li>O'Neill, J. S., Maywood, E. S., Chesham, J. E., Takahashi, J. S., Hastings, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP-dependent+signaling+as+a+core+component+of+the+mammalian+circadian+pacemaker.">cAMP-dependent signaling as a core component of the mammalian circadian pacemaker.</a> Science. 320, 949-953 (2008).</li><li>Hughes, A. T., Guilding, C., Lennox, L., Samuels, R. E., McMahon, D. G., Piggins, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+altered+period1+expression+in+the+suprachiasmatic+nuclei+of+Vipr2-/-+mice.">Live imaging of altered period1 expression in the suprachiasmatic nuclei of Vipr2-/- mice.</a> J Neurochem. 106, 1646-1657 (2008).</li><li>Zhang, D. Q., Zhou, T., Ruan, G. X., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+Period1+clock+gene+expression+in+NOS+amacrine+cells+of+the+mouse+retina.">Circadian rhythm of Period1 clock gene expression in NOS amacrine cells of the mouse retina.</a> Brain Res. 1050, 101-109 (2005).</li><li>Quintero, J. E., Kuhlman, S. J., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+clock+nucleus:+a+multiphasic+oscillator+network+regulated+by+light.">The biological clock nucleus: a multiphasic oscillator network regulated by light.</a> J Neurosci. 23, 8070-8076 (2003).</li><li>LeSauter, J., Yan, L., Vishnubhotla, B., Quintero, J. E., Kuhlman, S. J., McMahon, D. G., Silver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+half-life+GFP+mouse+model+for+analysis+of+suprachiasmatic+nucleus+organization.">A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization.</a> Brain Res. 964, 279-287 (2003).</li><li>Nakamura, W., Yamazaki, S., Takasu, N. N., Mishima, K., Block, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+Period+1+expression+within+the+suprachiasmatic+nucleus.">Differential response of Period 1 expression within the suprachiasmatic nucleus.</a> J Neurosci. 25, 5481-5487 (2005).</li><li>Davidson, A. J., Yamazaki, S., Arble, D. M., Menaker, M., Block, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+of+central+and+peripheral+circadian+oscillators+in+aged+rats.">Resetting of central and peripheral circadian oscillators in aged rats.</a> Neurobiol Aging. 29, 471-477 (2008).</li><li>Croning, M. D., Haddad, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+brain+slice+chamber+designs+for+investigations+of+oxygen+deprivation+in+vitro.">Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro.</a> J Neurosci Methods. 81, 103-111 (1998).</li><li>Burgoon, P. W., Boulant, 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=Temperature-sensitive+properties+of+rat+suprachiasmatic+nucleus+neurons.">Temperature-sensitive properties of rat suprachiasmatic nucleus neurons.</a> Am J Physiol Regul Integr Comp Physiol. 281, 706-715 (2001).</li><li>Nishide, S. Y., Honma, S., Honma, 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+circadian+pacemaker+in+the+cultured+suprachiasmatic+nucleus+from+pup+mice+is+highly+sensitive+to+external+perturbation.">The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation.</a> Eur J Neurosci. 27, 2686-2690 (2008).</li><li>Yoshikawa, T., Sellix, M., Pezuk, P., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+the+ovarian+circadian+clock+is+regulated+by+gonadotropins.">Timing of the ovarian circadian clock is regulated by gonadotropins.</a> Endocrinology. 150, 4338-4347 (2009).</li><li>Kohn, R. A., Dunlap, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+the+buffering+capacity+of+bicarbonate+in+the+rumen+and+in+vitro.">Calculation of the buffering capacity of bicarbonate in the rumen and in vitro.</a> J Anim Sci. 76, 1702-1709 (1998).</li><li>Wilsbacher, L. D., Yamazaki, S., Herzog, E. D., Song, E. J., Radcliffe, L. A., Abe, M., Block, G., Spitznagel, E., Menaker, M., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photic+and+circadian+expression+of+luciferase+in+mPeriod1-luc+transgenic+mice+in+vivo.">Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo.</a> Proc Natl Acad Sci U S A. 99, 489-494 (2002).</li><li>Sellix, M. T., Currie, J., Menaker, M., Wijnen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence/luminescence+circadian+imaging+of+complex+tissues+at+single-cell+resolution.">Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.</a> J Biol Rhythms. 25, 228-232 (2010).</li></ol>

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ルシフェラーゼレポーターの技術を持つメリットとデメリット昼間の勉強するために多くの異なる時点（サンプリング周波数に応じて、通常2-4時間の低時間分解能を与える）で組織採取を必要とするin situハイブリダイゼーション及びウエスタンブロットで 、このようなRT - PCRなどのex vivoでの方法とは対照的に、遺伝子と蛋白質の表現の変化、ルシフェラーゼレポーター技術は、高解像度（1-10分）と同じ準備で何日も概日振動の研究をすることができます。したがって、使用する動物の数は最小限に抑えられ、リズムの位相と周期への影響の詳細な研究は、通常、低時間分解能と、従来のサンプリング技術を利用可能ではない、実現可能である。しかし、リズムの相対的な振幅の測定が可能であるが、それはレポーター技術は定量的でないため、転写された遺伝子または翻訳されたタンパク質の量を測定するために使用することができないことが強調されるべきである。 組織の記録は、器官SCNの文化は成人の脳の長期的な（週）薬理学的操作を可能に開始し、直ちに培養、直接生体内光誘起位相シフトなどで 、 生体のストレスでの分子の影響を研究するための大きな利点の後に分析することができます無傷の成熟したシナプスのネットワークとルシフェラーゼレポーターの技術を含む組織は、何週間も安定した記録が可能になります。したがって、組織が健全な文化を得るために新生児または出生後早期にする必要はありません、と急性スライス慢性の薬理学的治療とは対照的に行うことができます。エピジェネティックな変化が検討される場合はないゲノムへのDNAの取り込みにつながるか細胞培養におけるプラスミド - レポータートランスフェクション、とは対照的に、トランスジェニックルシフェラーゼ - 動物モデル（だけでなく、レンティ-ウイルスのトランスフェクション）が良好である。それはこの文脈でも発光イメージングは、レコーディングにも概日リズムを研究の主要な研究室によって利用される単一のSCNのニューロン（〜60から10ミクロン）や他の細胞型、許されるように、高感度CCDカメラ11で、今日可能であることが述べられるべきである。最後に、いくつかの概日研究者は、一般的に時計遺伝子/蛋白質の振動16-19を勉強するために、そのような緑色蛍光タンパク質として、他のレポーターを用いた分子の発現のモニタリングを使用してください。 スライス厚の側面必要に応じて、ここで推奨SCNのスライス（200〜300ミクロン）の厚さが減少または増加させることができた。組織の文化の中で数日後に膜に平坦化するが、しかし、、それは組織7の実行可能性を維持するために最初にカットスライスの500μmの厚さを超えることは推奨されません。が100μmよりも薄いスライスは取り扱いが困難機械的および実際的な理由からです。 SCNの組織が不均質であるため、SCN（例えば、&quot;シェル&quot;と&quot;コア&quot;、と背比べ腹側の領域）内の異なる領域が異なる位相で振動するので、スライスの厚さは、ルシフェラーゼの出力信号の位相に影響を及ぼす可能性がありますフェーズの後に20と差動再同期は、21をシフト。より多くの組織は、光子の大きい数を発するので、スライスの厚さも光子数のベースラインに影響を与えます。分子振動の振幅は、このように間接的に植の大きさによって影響を受ける可能性があります。これらの理由から、制御と処理培養物は、常に同じ大きさ、厚さであることと同じ位相で、同じ振幅で発振するために、SCNの同じ領域を含む必要があります。 two一方的な核は、その一方で、ビブラトームカットが順番に小脳と二つの半球の間に切断分離が垂直であることに依存している水平方向と（角度がない限り、お互いにと同様の振幅と位相で振動するテーブルの表面に）。 SCNの培養を行う研究者は、SCNの文化は互いに位相で発振しないことが発生する可能性があります。練習とスライシング技術の標準化、スライスは常にSCNの同じrostro -尾レベルでカットされている、すなわち、結果が改善されます。 重要なステップ<ol><li>酸素供給は重要です。 脳は酸素22の多くを必要とするため、スライシング手続きは迅速である必要がある（3.1から3.10に近い、より良い、それが組織を健康に保つためにある〜5-6分です）。 </li><li> SCNは、温度変化や培地交換に敏感です。 SCNは、温度に敏感であり、大きな温度変化は、位相ペースメーカーをシフトし、実験的なアーティファクト23を引き起こす可能性があります。培地やソリューションは、培養中の数日後に薬を適用するなどの継続的な実験、、培地またはsolutio中に交換または追加される場合nは、さらに前に36〜37 ° C（チャンバー内と同じ温度）に事前に温める必要があります。培地交換を伴う薬理学的実験では、それは常に事前にエアコンの細胞培養培地と連携することが重要です。さらに/新鮮な、無条件の培地でアウト洗浄は、潜在的に分子のSCNのリズム24だけでなく、他の細胞型の培養25を位相シフトすることができます。 </li><li>培地組成物。 正しいpH及び浸透圧の文化組織をすることが重要です。空気緩衝培地はpHが6.8から8.2の範囲で最適なバッファ容量を持ち、細胞の呼吸の結果として発生する可能性のあるpHの変化をバッファリングするためにも適切であるHEPESが大量に含まれています。 HEPESは、一方、として培地中に少量に含まれている炭酸水素ナトリウム、と比較して温度変化に対して敏感です。重炭酸ナトリウムは、CO 2雰囲気下でこのように適切な低いpH（5.1〜7.1）範囲26内のより大きな緩衝能力を持っています。しかし、空気緩衝培地（DMEM 2902）に重炭酸ナトリウムの量を増加が大幅に浸透圧を増加させると同時に培地、アミノ酸、ビタミンおよびイオンの不正確な組成のターン結果に含めます。希釈することなく追加することができます。 培地を調製するためのアドバイス： <ol><li>媒体とを混合する際、正確なと注意してください。 </li><li>だけ新鮮か新鮮解凍したストック溶液を使用してください。 </li><li>非常に高い浸透圧の場合には、希釈する価値はありません。以上の5から6パーセントに希釈した培地は、ほとんどの場合動作しません。それが低すぎる場合浸透圧を高めるために、D -グルコースを使用してください。 </li></ol></li><li>フェーズは準備時間に起因するシフト スライス標本の時間は非常に重要です。スライスの手順はZT 6月12日12の間に実行された場合、位相でゼロまたは最小限の効果が得られる。すべてのスライスの解離は、準備の間の位相変動に起因する誤差を最小化するために、日周または概日周期の同位相で実行する必要があります。 </li></ol>実行可​​能な変更<ol><li>スライスが開始する前に酸素（95％O 2、5％CO 2）で飽和バッファー、急性SCNの電気生理学の場合、スライスはリンガー（ACSF人工脳脊髄液）で実行する必要があります。さらに、酸素は、電気とシナプス活動は、酸素の電源22に直接依存しているとして、全体のスライス手順の実行中にバッファに連続して追加する必要があります。 </li><li>水平方向と矢状スライスは正常に準備し、SCNを培養することができる。著者は、我々の経験によるとコロナのカットと比較して低い振幅で概日振動を与える水平スライスにおけるルシフェラーゼの録音と経験を、持っている。 </li><li> SCNはrostro - 尾側に長さ約1mmであるとスライシング技術は、ここで説明する中央部にSCNのセクションを取得を対象としています。ただし、複数のSCNのセクションは、SCNの核のより吻側対尾側レベルでの概日遺伝子/タンパク質発現の調査を可能にする、マウスおよびラットの脳から得ることができる。 </li><li>さらに、若い動物、出生後の仔や胚からスライスの培養物は、また、調製することができる。非常に若い子犬の脳と頭蓋骨は非常に柔らかく、敏感であることに注意してください。さらに、視神経は薄いと十分に発達していないです。 SCNの領域が損傷していないように仔の脳を解剖する際はご注意ください。彼らは大きすぎるので、例えば、マイクロ骨鉗子のツールは、仔マウスの頭蓋骨を開くために使用することができます。ファインはさみは若い仔で軟部組織を切断するのに十分です。 </li><li>ここで説明するカバーガラスベースの培養技術は、単一SCNのニューロンでは、CCD / EM - CCDカメラを用いた画像の発光にも使用できます。 </li><li>他の中枢神経系領域とトランスジェニックPER2から末梢器官組織：：LUCの動物が容易にPER2として、分子振動の面で、得られる培養し、分析することができます：：LUCの発現が他の組織の数にも多くの日のために発振し続けると細胞のタイプ10。これらの末梢組織のほとんどは、インスタンスの肝臓のために、生存のための膜を必要としないのではなく、直接媒体11を浴びて培養されています。により、切片にシフトその位相を注意してください、培養と培地交換はSCNと同じ原則に従っていない可能性があります。 </li></ol>意義トランスジェニックマウスとラット系統（：mPER2：LUC、mPer1 - lucを8、10、27）でルシフェラーゼ酵素活性は、タンパク質や遺伝子発現のリズムを反映し、生物発光の記録によって評価することができる。ルシフェラーゼによって生成される生物発光は、微弱な信号を与えるが、バックグラウンド発光は、この方法が有利なこと、ゼロに近いです。また、ルシフェラーゼ分子は不安定で速やかに分解されるためにはpHはありません長期的な興奮照明28の間に表示される可能性難聴、。一緒に、これらのプロパティは、概日研究において非常に有利なルシフェラーゼレポーターの技術を作る長期的な実験を可能にする。

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<tr>
<td>B27 supplement 50x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Cover glasses</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser</td>
<td>40#1</td>
<td>&#8776; 40 mm</td>
</tr>
<tr>
<td>Culture membranes</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG50</td>
<td>0.4 &mu;m</td>
</tr>
<tr>
<td>DMEM low glucose w/o phenol red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2902</td>
<td>Powder for 1L</td>
</tr>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1003 055</td>
<td>&#8776; 55 mm</td>
</tr>
<tr>
<td>Filtration set</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>431097</td>
<td>Pore size 0.22 &mu;m
Polyethersulfone</td>
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<td>Forceps </td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>0203-7-PS</td>
<td>Dumant #7</td>
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<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15630-056</td>
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<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco </td>
<td>14065-049</td>
<td>500 ml</td>
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<td>NaOCH3 7.5%</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25080-060</td>
<td>50 ml</td>
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<td>Luciferin </td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1602</td>
<td>Beetle luciferin, potassium salt</td>
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<td>Micro rongeur tool</td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>332-097-140</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep 10,000 U/ml</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15140-122</td>
<td>100 ml</td>
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<td>&nbsp;</td>
<td>Corning</td>
<td>430165</td>
<td>&#8776; 35 mm</td>
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<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>H9319-11MOD</td>
<td>&nbsp;</td>
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<td>Disposable scalpels</td>
<td>&nbsp;</td>
<td>Paragon</td>
<td>P503</td>
<td>Size 11</td>
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<tr>
<td>Vacuum filter</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>09-761-5</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>50 g</td>
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<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Campden Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus

生理学と行動で毎日のリズムを調整する中心的な概日リズム（〜24時間）クロックは、視床下部前部にある視交叉上核（SCN）に存在する。クロックは、直接網膜と視神経を介して光によって同期化されます。概日振動は、 周期 （ 単位 ）の遺伝子を含む、いわゆる&quot;時計遺伝子&quot;と、そのタンパク質産物の数、負のフィードバックループを相互作用によって生成されます。コアクロックはまた、膜の脱分極、カルシウムやキャンプ1に依存しています。 SCNは、時計遺伝子の発現、代謝活性と自発電気活動の毎日の振動を示しています。驚くべきことに、この内因性の周期的活動は、SCN 2-4の成体組織スライスに保持されます。この方法では、体内時計は簡単にペースメーカー機能の分子、電気生理学的及び代謝の研究を可能にする、in vitroで研究することができます。 SCNは、右の視交叉5の上方に位置する小規模な、よく定義された二国間の構造体です。ラットでは、それぞれの核に〜8.000ニューロンが含まれており、約947ミクロン（長さ、rostrocaudal軸）× 424μmの（幅）× 390μmの（高さ）6の寸法を有する。それは、SCNを識別することができる脳の特定のレベルでの脳のスライスをカットするために必要なSCNを分析する。ここで、我々は、マウスやラットの脳についても同様であるSCN、の解剖とスライスの手順を説明します。さらに、我々はどのように膜7、山崎らによってSCNの組織培養用に開発された技術上organotypically解剖組織培養に示す。8。最後に、我々は、動的なルシフェラーゼレポーターテクノロジー、もともとGeusz ら概測定に使用されたメソッドを使用して時計遺伝子/タンパク質の発現を測定するために使用する方法をトランスジェニック組織を示しています。9。 ：我々はトランスジェニックノックインPERIOD2からSCN組織をここでを使用してください：。ユジンらが生成されるルシフェラーゼマウス10。マウスは、PERIOD（PER）の融合タンパク質を含んで2とホタルの酵素ルシフェラーゼ。ルシフェラーゼは、ルシフェリンの酸化を触媒するときにルシフェラーゼの基質の存在下で翻訳されている場合PER2、すなわちルシフェリン、PER2の発現を生物発光として監視することができます。放出されるフォトンの数は、積極的に生産さPER2蛋白質の量に相関し、そして生物発光リズムは生体 10 で PER2蛋白質のリズムと一致している。この方法ではPER2の発現の周期的な変動が継続的に多くの日中にリアルタイムに監視することができます。我々は組織培養とリアルタイムの生物発光のレコーディングに従うプロトコルは、徹底的に山崎と高橋11で記載されている。

Watch this Scientific Journal Video about Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus at JoVE.com

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

成体マウス視床下部視交叉上核（SCN）、そして文化への迅速な方法器官培養条件下でのSCNの組織を含む準備スライス、の手順が報告されています。さらに、動的なルシフェラーゼレポーターの技術を使用して振動時計遺伝子のタンパク質発現の測定が記述されています。

スライスの準備、器官組織培養と視交叉上核における時計遺伝子の活動のルシフェラーゼ記録

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

slice-preparation-organotypic-tissue-culturing-luciferase-recording

Research

JoVE Journal

Neuroscience

34.9K ビュー.  Karolinska Institutet. 成体マウス視床下部視交叉上核（SCN）、そして文化への迅速な方法器官培養条件下でのSCNの組織を含む準備スライス、の手順が報告されています。さらに、動的なルシフェラーゼレポーターの技術を使用して振動時計遺伝子のタンパク質発現の測定が記述されています。

ビデオ: Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神経科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

新皮質の器官型スライス標本からの全細胞記録

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

マウス鋤鼻急性組織スライス標本のイメージングフェロモンセンシング

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

器官切片培養におけるプルキンエ細胞の樹状突起の形態分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Entorhino海馬器官型スライス培養における神経血管リモデリングの解析

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.