Name: recording gamma band oscillations in pedunculopontine nucleus neurons
Uploaded: 2016-09-14T15:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

全ての実験プロトコルは、アーカンソー医科大学の施設内動物管理使用委員会（プロトコル番号＃3593）によって承認され、実験動物の管理と使用のための国立衛生研究所のガイドラインと一致したしました。 標準人工脳脊髄液（aCSFの）の調製<ol><li> 原液Aの調製 <ol><li>化学物質を添加する前に、きれいな1リットルのビーカーに蒸留水700ミリリットルを追加します。 </li><li>連続して500ミリリットルの容量を攪拌しながら、塩化ナトリウムの136.75グラム、塩化カリウムの6.99グラム、 硫酸マグネシウムの2.89グラム、およびのNaH 2 PO 4の2.83グラムを追加します。 </li><li>希釈した後、1リットルの最終体積に到達するより多くの蒸留水を追加し、各化合物の最終濃度は、117 mMの塩化ナトリウム、4.69ミリモルのKCl、1.2mMのMgSO 4を、および1.18ミリモルのNaH 2 PO 4です。 </li><li> 2週間まで4℃で冷蔵して保管する。 </li></ol></li><li> ストック溶液Bの調製 <ol><li>化学物質を追加する前にクリーン1Lビーカーに蒸留水700ミリリットルを追加します。 </li><li>連続して500ミリリットルの容量を攪拌しながら、D-グルコースおよびNaHCO 3の41.84グラムの41.45グラムを追加します。 </li><li>希釈した後、1リットルの最終体積に到達するために多くの蒸留水を追加し、各化合物の最終濃度は11.5 mMのD-グルコース、および24.9 mMの炭酸水素ナトリウムです。 </li><li> 2週間まで4℃で冷蔵して保管する。 </li><li> 50ミリリットルストックBと株式Aの各実験ミックス50ミリリットルの前に、最終濃度aCSFの溶液を得、継続的にカルボゲンでそれを酸素化しながら、室温で残すように蒸留水で1 Lにもたらす（95％O 2から5パーセント CO 2ミックス）少なくとも30分間。のみ各実験の開始時に最終濃度aCSFのを準備し、一日の終わりに捨てます。 </li></ol></li></ol>スクロース、人工脳脊髄液の調製（スクロース-aCSFの） <ol><li> ストック溶液 C の調製 <ol><li>化学物質を添加する前に、きれいな1リットルのビーカーに蒸留水700ミリリットルを追加します。 </li><li>連続蒸留水500mlを攪拌しながら、ショ糖の240グラム、 炭酸水素ナトリウムの6.55グラム、塩化カリウムの0.671グラム、MgCl 2を4.88グラム、 塩化カルシウムの0.22グラムとアスコルビン酸の0.21グラムを追加します。 </li><li>希釈した後、1リットルの最終体積に到達するより多くの蒸留水を追加し、各化合物の最終濃度は701.1 mMのスクロースであり、78ミリモルのNaHCO 3、9ミリモルのKCl、24ミリモルのMgCl 2、1.5mMのCaCl 2を1.2 mMのアスコルビン酸。 </li><li> 1週間まで4℃でストック溶液Cを保管してください。 </li><li>各実験ミックス前に連続的にカルボゲン（95％O 2 - 5％CO 2混合物）を用いてそれを酸素化しながら、最終濃度スクロースaCSFの溶液を得、室温で残すように蒸留水600mlでストックCを50mlを300ml少なくとも30分間。 </li></oL&gt; </li></ol> 3.スライスの準備<ol><li>カルボゲンでそれを酸素化しながら、氷で100ミリリットルスクロースaCSFの溶液できれいなビーカーを置き、0.1 M NaOH溶液数滴を加えながらpHメーターを用いて7.4にpH値を固定する（ときのpH &lt;7.4）または0.1 M HCl溶液（ pHが&gt; 7.4）。 </li><li>ショ糖aCSFのでビブラトームのカッティング室を埋めると、それに酸素。カッティングチャンバに結合されたグリセロールベースの​​冷凍システムの電源をオンにし、それが0に冷却できるようにするために15分を待つ - 4°C。 </li><li> （; &lt;50μlの最終容量を使用して、70 mgの/ kg、 腹腔内）ケタミンと（大人時限妊娠Sprague-Dawleyラットから12に9歳）仔ラットを麻酔。テールピンチ反射が存在しないことを子犬が穏やかで、ダブルチェック。 </li><li>子犬を首を切ります。 <ol><li>炭素鋼メスの刃を使用して、前から後ろに縦方向に頭部の皮をカットし、鉗子を使用して、側面に皮膚を引っ張ります。皮下に移動脳を覆っている骨をカット横方向にissorsと完全に脳を露出するために、それを削除します。 </li><li>その後、急速に（最初は静かに最も尾側の領域に向かって最も吻側から脳を押し出すために、嗅球下に置かれた）スパチュラを用いて脳を削除します。静かに氷冷した酸素スクロース人工脳脊髄液（ショ糖aCSFの）に脳を押してください。 </li></ol></li><li> （半球の約3分の1を除去すること）右半球上の矢状カットを行い、磁気含む矢状400μmのセクションをカットするvibroslicerの切断室に固定される金属製のディスクに脳のトリミングされた側を接着脚橋核（PPN）。全細胞パッチクランプ記録に先立って室温で45分間PPNスライスにしてください。 </li></ol> PPNスライス4.録音ガンマ帯振動<ol><li> カリウム、グルコン酸細胞内液の調製（高K +溶液） <ol><li>挟み込みます氷10ミリリットルの蒸留水できれいなビーカー。連続して撹拌しながら追加：K + -gluconate、90.68 mgのクレアチンリン酸ジトリス塩、47.66ミリグラムのHEPES、1.5ミリグラムのEGTA、40.58ミリグラムのMg 2+ -ATP、および4 mgののNa + 2 -GTPを。 </li><li> KOH（蒸留水中の100mM）でpHを7.3に調整します。 20 mLの最終体積に達するように蒸留水を加えます。 290 mOsmで - 必要な場合は、280であることが、スクロース（蒸留水中の100mM）との浸透圧を調整します。希釈した後、各化合物の最終濃度は、124 mMのK + -gluconate、10mMのクレアチンリン酸ジトリス塩、10mMのHEPES、0.2mMのEGTA、4mMののMg 2+ -ATP、および0.3mMのナトリウム+ 2 -GTPあります。 </li><li>一定分量の細胞内液1ミリリットル試験管内および-20℃で凍結します。一日あたりの1つのアリコートを使用し、実験中に4℃で維持。 </li></ol></li><li> 全細胞パッチクランプ記録 <ol><li>浸漬室にスライスを配置し、酸素とのそれら（1.5ミリリットル/分）を灌流（95％O 2 - CO 2 5％）以下の受容体アンタゴニストを含有するaCSFの：選択的NMDA受容体アンタゴニスト2-アミノ-5-ホスホノ吉草酸（APV、40μM）を、競合的AMPA /カイニン酸グルタミン酸受容体アンタゴニスト、6-シアノ-7- nitroquinoxaline -2,3-ジオン（CNQX、10μM）、グリシン受容体アンタゴニストストリキニーネ（STR、10μM）、特定のGABA受容体アンタゴニストgabazine（GBZ、10μM）、およびナトリウムチャネルブロッカーテトロドトキシン（TTX、3μM） 注：結果と図では、これらのアンタゴニストをまとめてシナプスのブロッカーまたはSBSと呼ばれています。 </li><li>市販のパッチピペットフィラーを使用して、細胞内の高K +溶液で、（1.0ミリメートル外径0.6ミリメートル、内径の定期的な、市販の肉厚ホウケイ酸ガラスキャピラリーから作られた抵抗2-7MΩ）記録パッチピペットを埋めますソリューションフィルター。そのアンプのホルダーにピペットを挿入します。小さなPOSを適用します三方弁に接続されたシリコンチューブを用いたピペットホルダーに接続された1mlシリンジを用いitive圧力。パッチクランプアンプにピペットホルダーの背面に接続します。 </li><li>近赤外微分干渉コントラスト光学系に合わせた4Xの対物レンズを使用してPPN核の近くに機械的なマイクロマニピュレーターを用いた記録ピペットを移動します。 注：（;軸索の厚い束として観察可能なSCP）PPN核は、上小脳脚にスライスの背側で観察することができます。記録ピペットは、PPN内に配置された直後に花柄の後端に背側に配置されて緻密に 、 扁平部 。 </li><li> 40X水浸レンズを用いて可視化しながら、PPNニューロンと接触して記録ピペットを持参し、急速に細胞とのシールを形成するために、負の吸引を適用します。 </li><li>製造業者のプロトコルを使用して負の吸引時にピペット抵抗を監視するために電圧クランプシールソフトウェアを使用してください。 <ol><li>ときに負のuctionが徐々に増加し、ピペットの先端のパッチクランプモニタによって読み出した抵抗値が80に達する - 急速mVの-50負圧を解放する保持電位を変更する、100オームです。ニューロンの膜を破裂し、電気的アクセスが全細胞構成で達成されるまで負の吸引を適用する継続的に起動します。 </li><li>電圧クランプシールソフトウェアによって測定さアクセス抵抗値が10オーム以上であれば、少量で負の吸引を適用し続けます。 </li></ol></li><li>静電容量を補償する電圧クランプモードおよび直列抵抗（ すなわち 、低速および高速過渡現象は、細胞の膜を破壊した後に観察されました）。ブリッジ値を補償急速に電流クランプに録音モードを切り替えて、（ 例えば 、アンプのつまみを移動したり、computer&#39;sマウスを使用して自動補正]メニューをクリックしてください）。 <ol><li>連続uと記録されているPPNニューロンの静止膜電位監視製造業者のプロトコルを歌います。脱分極または過分極の値に向かって膜電位のシフトを休んであれば、最適な-50 mVの最終値を維持するために直流電流（最大100 PA）の少量を使用します。 注：-48 mVでの安定した静止膜電位（RMP）以上の過分極（ すなわち 、RMP &lt;-48 mVの）でのみPPNニューロンを保管してください。 </li></ol></li></ol></li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+neurons+in+the+dorsolateral+pontine+REM+sleep+induction+zone+of+the+rat:+intrinsic+membrane+properties+and+responses+to+carbachol+and+orexins.">Electrophysiological characterization of neurons in the dorsolateral pontine REM sleep induction zone of the rat: intrinsic membrane properties and responses to carbachol and orexins.</a> Neuroscience. 143 (3), 739-755 (2006).</li><li>Goetz, L., 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=On+the+role+of+the+pedunculopontine+nucleus+and+mesencephalic+reticular+formation+in+locomotion+in+non-human+primates.">On the role of the pedunculopontine nucleus and mesencephalic reticular formation in locomotion in non-human primates.</a> J. Neurosci. 36 (18), 4917-4929 (2016).</li><li>Fraix, 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=Pedunculopontine+nucleus+area+oscillations+during+stance,+stepping+and+freezing+in+Parkinson's+disease.">Pedunculopontine nucleus area oscillations during stance, stepping and freezing in Parkinson's disease.</a> PLOS ONE. 8 (12), e83919 (2013).</li><li>Luster, B., Hyde, J., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">Mechanisms behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Abstr Soc Neurosci. 38, 257.20 (2014).</li><li>Luster, B., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-threshold+Ca2++channels+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Physiol. Rep. 3 (6), e12431 (2015).</li></ol>

The authors declare that they have no competing financial interests.

PPNニューロンは、彼らが目を覚ましやレム睡眠時ではなく、徐波睡眠2,3,5,13-17中にある動物からのin vivoでの録音中にベータ/ガンマ帯域周波数での活動電位を発射することを可能にする固有の特性を持っています。他の著者は、PPNよりも前方のレベルで脳幹離断は、EEG記録時の​​ガンマ周波数を低下させることが示されたています。しかし、脳幹病変は、この核が配置されて...

PPN核は解剖学的に尾側中脳被蓋に含まれています。 PPNは、RAS 1の主要コンポーネントです。 PPNは、行動活性化された状態（ すなわち 、覚醒、レム睡眠）2のメンテナンスに参加しています。ラットにおける二国間PPN病変が減少またはレム睡眠4を排除しながら、皮質脳波3に- （40 Hzの20）in vivoでの PPNの電気刺激は、高速発振を誘発しました。 PPNニューロンの大部分はベータ/ガンマ帯域周波数での活動電位発火しながら（20から80 Hz）を、いくつかのニューロンが自然発火の低金利を提示した（&lt;10 Hz）で5。また、PPNは、そのような動機と注意6のような行動の他の側面に関与しているようです。直接高周波（40から60 Hz）で、除脳動物でのPPN核の7電気刺激は、運動を促進することができます。近年、脳深部刺激（DBS）は、PPNのあちこちに罹患している患者を治療するために使用されていますパーキンソン病（PD）8と歩行障害を伴うM障害。 以前の報告は、方形波電流パルス9を使用して脱分極時にほぼすべてのPPNニューロンは、ガンマ帯域周波数での活動電位を発射することができることを実証しました。そのためmVのまでまたは-25下の矩形パルス脱分極時の電位依存性カリウムチャネルの急激な活性化。その結果、何の堅牢なガンマ振動はテトロドトキシン10を使用して、活動電位の生成を阻止した後に観察されませんでした。このような問題を回避するための努力では、1 - 長い現在のランプを脱分極2秒を使用しました。部分的に電位依存性カリウムチャネルを不活性化しながら、ランプは徐々に、0 mVの最大値を休んでから、膜電位を脱分極しました。透明なガンマ帯膜振動が10（-25 mVのと-0 mVの間、 すなわち、）高閾値カルシウムチャネルの電圧依存性ウィンドウ内に明らかでした。結論として、ガンマバンドactivityがPPNニューロン9において観察され、両方のPは/ Q-とN型電位依存性カルシウムチャネルは、PPN 10ガンマ帯の振動を生成するために活性化される必要があります。 一連の研究は、PPNニューロンにおける高閾値カルシウムチャネルの位置を決定しました。染料の組合せを注入し、 レシオメトリック蛍光イメージングは、現在のランプ11を使用して脱分極時に別の樹状突起で活性化された電位依存性カルシウムチャネルを介してカルシウム過渡応答を示しました。 PPNニューロンの固有の特性は、このようにRASと視床皮質ループ間に高周波振動神経活動を誘導し、覚醒およびレム睡眠中に、これらの細胞の同時活性化を可能にするために提案されています。このような長期に及ぶ相互作用を確実に継続的12上で私たちの周りの世界を評価することのできる脳の状態をサポートするために考えられています。ここでは、実験を記述する必要アル条件が生成し、in vitroで PPN細胞におけるガンマ帯発振を維持します。このプロトコルは、以前に記載されておらず、他の脳領域でガンマバンド活性を媒介する固有の膜特性を研究するグループの数を助けます。また、現在のステップは、ガンマバンド活動がこれらの細胞内で生成することができないという誤った結論につながる可能性があります。

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;, molecular weight = 342.30</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 84.01</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td>KCl, molecular weight = 74.55</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =&amp;nbsp; 203.30</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =147.02</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight = 180.16</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight =176.12</td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td>NaCl, molecular weight =&amp;nbsp; 58.44</td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;KO&lt;sub&gt;7&lt;/sub&gt;, molecular weight =&amp;nbsp; 234.25</td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td>C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;P &amp;middot; 2C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, molecular weight =&amp;nbsp; 453.38</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S, molecular weight = 238.30</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>[-CH&lt;sub&gt;2&lt;/sub&gt;OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;N(CH&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H)&lt;sub&gt;2&lt;/sub&gt;]&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 380.40</td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td>&amp;nbsp;C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;13&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xMg2+, molecular weight = 507.18</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;14&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xNa+, molecular weight = 523.18</td></tr><tr><td>Tetrodotoxin citrate</td><td>Alomone Labs</td><td>T-550</td><td>C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;, molecular weight = 319.27</td></tr><tr><td>&amp;nbsp;DL-2-Amino-5-Phosphonovaleric Acid</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P, molecular weight = 197.13</td></tr><tr><td>CNQX disodium salt hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C239</td><td>C&lt;sub&gt;9&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt; &amp;middot; xH2O, molecular weight = 276.12</td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>C&lt;sub&gt;21&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 334.41</td></tr><tr><td>Mecamylamine hydrochloride</td><td>Sigma-Aldrich</td><td>M9020</td><td>&amp;nbsp;C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;21&lt;/sub&gt;N &amp;middot; HCl, molecular weight = 203.75</td></tr><tr><td>Gabazine (SR-95531)</td><td>Sigma-Aldrich</td><td>S106</td><td>C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;BrN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 368.23</td></tr><tr><td>Ketamine hydrochloride</td><td>Mylan</td><td>67457-001-00</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>Pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>&amp;nbsp;Leica VT1200 S</td><td></td></tr><tr><td>Refrigeration system</td><td>Vibratome Instruments</td><td>900R</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr></tbody></table>

recording gamma band oscillations in pedunculopontine nucleus neurons

（; CL / Pfの核例えば 、centrolateral / parafascicular）PPNからシナプス遠心性神経は、いくつかの髄板内視床領域の神経活動を調節することが知られています。 PPNまたはインビボでのCl / Pfの核のいずれかの活性化は、動物の覚醒および皮質脳波（EEG）ガンマ帯活性の増加を誘導することが記載されています。網様体賦活系（RAS）ニューロンにおけるガンマ帯の振動の発生のための細胞機構は、他の脳核ガンマ帯振動を発生することが判明したものと同じです。 （9からの矢状スライスから - 25日齢ラット）PPNニューロンの電流クランプ記録時には、正方形の手順を脱分極の使用が急速に-25 mVで越えて脱分極されることからPPNニューロンを防ぐ電位依存性カリウムチャネルを活性化しました。 1注入 - 長い現在のランプを脱分極2秒と、徐々にPPNの膜電位解像度を脱分極しましたティンは0 mVで向かっ値。しかし、注入脱分極方形パルスはランプによって生成される振動に比べて振幅が小さいことが示された膜電位のガンマ帯振動を発生します。全ての実験は、電位依存性ナトリウムチャネル及び速いシナプス受容体遮断薬の存在下で実施しました。高閾値電圧依存性カルシウムチャネルの活性化は、PPNニューロンにおけるガンマ帯振動活動の根底にあることが示されています。具体的な方法論的および薬理学的介入は、in vitroで PPNサブスレッショルドガンマ帯振動を誘発し、維持するために必要なツールを提供し、ここで説明されています。

最初は、ガンマ振動は方形波電流パルスを用いて誘発されました。シナプスのブロッカーとTTXの存在下でPPNニューロンの電流クランプ記録を連続静止膜電位は〜-50 mVの（ 図1A）で安定に保持したことを保証するために監視しました。二つの第二の長い方形電流パルスは200 PAから600 PA（ 図1A）への振幅を増加させる、記録ピペットを介して、パ...

Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

脚橋核（PPN）は脳幹に位置し、そのニューロンが最大限に起床し、急速眼球運動（REM）睡眠脳の状態の間に活性化されています。この作品は、PPNニューロンのin vitroガンマバンドサブスレッショルド膜振動に記録する実験的なアプローチについて説明します。

脚橋核ニューロンにガンマバンド振動を記録

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

recording-gamma-band-oscillations-in-pedunculopontine-nucleus-neurons

Research

JoVE Journal

Neuroscience

8.6K ビュー.  University of Buenos Aires. 脚橋核（PPN）は脳幹に位置し、そのニューロンが最大限に起床し、急速眼球運動（REM）睡眠脳の状態の間に活性化されています。この作品は、PPNニューロンのin vitroガンマバンドサブスレッショルド膜振動に記録する実験的なアプローチについて説明します。 

ビデオ: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

運動によって変調された神経細胞の生理的、形態学的および神経化学的キャラクタリゼーション

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

神経科学

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

マウス脳波で高度に組織化されたシータ振動の自動検出

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

行動学

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

用ピギーバックMultibarrel電極の製造と使用<emインビボ&gt;</em神経反応の&gt;薬理マニピュレーション

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

テタヌス刺激による局所CA1γ振動の発生

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

海馬シータバンドのチューニング<em&gt;インビトロ</em&gt;：隔離されたげっ歯類胸腺腫回路からの記録方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;インビボ</em&gt;麻酔ラットにおけるHyperdirect経路からの局所場ポテンシャルとマルチユニット活性の電気生理学的記録

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

行動するマウスにおける海馬シータ振動の光同調

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行動中マウスの海馬における振動を制限空間録音

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

網膜原生殖器およびコルチコゲン化シナプス機能の電気生理学的調査

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Source: Urbano, F. J., et al. <a href="https://app.jove.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

脳スライスにおける小児性核ニューロンからのガンマバンド振動の記録

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

脚橋核ニューロンにガンマバンド振動を記録

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