この作品は、健康のインディアナ国務省から脊髄や脳傷害研究資金からNIH / NINDS助成4R00 NS 057940、および助成金SCBI 200から12でサポートされていました。

1。アンダーカット手術のためのシンプルなデバイスを作る<ol><li>我々が作成したアンダーカット装置は三つの部分（図1）で構成されています：誘導管の添付ファイルと針を可能にし、手術中に頭蓋窓を介して座っているステンレススチールまたはプラスチック製（1）支持板（2）位置および許可の針の回転で針を保持する誘導管、および先端から約3 mmに90度曲がっている（3）針、および回転および挿入によって移動可能である。 </li><li>支持板を作るために厚さ1〜1.5ミリメートルの一部、7〜10 × 30ミリメートルの長方形ステンレス製または透明プラスチック（1）カット。 1.5インチ22ゲージ（BD会社、＃305156）と1.5インチの25ゲージ（BD会社、＃305127）シリンジの針を準備します。誘導管を作るために、プラスチックの端とその合計の長さが31〜32ミリメートル（2）についてになるように22ゲージの注射針の下針の先端を切断。砂、金属の端を、それが平らで滑らかにする。この誘導管の最後の長さは25ゲージの注射針に比べ約5〜6ミリメートル短くしてください。 </li><li>針は、金属のエッジに対して垂直であることを確認し、支持板の上に誘導管を固定するシアノアクリレート接着剤を使用してください。 </li><li>誘導管に25ゲージ注射針を挿入し、先端（図1）2.5〜3ミリメートルで針を90度曲げる。 </li><li>針の上の端（図1、針の停止）に接着する小さなプラスチック製のチューブで針の垂直方向の移動範囲を調整します。針の最終的な移動範囲は、針を皮質に挿入する方法を深く決定し、P21ラットとP21マウスの1.3〜1.5ミリメートルのための1.6〜1.8ミリメートルでなければなりません。この長さは、異なる種や動物の年齢のために調整する必要があります。 </li><li>曲がった先端（図1針の指標）と同じ方向に針の上端に小さな銅線またはテープの小片を取り付けます。ワイヤーやテープは、アンダーカット手術（図1）中の針の回転角度を示します。 </li></ol> 2。動物の準備<ol><li>すべての手術器具は、ガラスビーズ滅菌器、または70％エタノールで消毒と滅菌、オートクレーブする必要があります。 </li><li>同じ年齢で22日間（P20 - 22）またはCD1マウスに - 私たちは、出生後20歳でのSprague / Dawleyラットを使用してください。マウスやラットの異なる株は、特定のプロジェクトのために使用することができます。 </li><li>ケタミン80 mg / kgとキシラジン8 mg / kgの腹腔内注射したマウスを麻酔。動物は、尾のピンチに応答しない後に手術が開始されます。手術中は、必要に応じて、麻酔薬のカクテルの三分の一元用量のブースター投与量は、元の麻酔の状態を復元するために与えることができます。 </li><li>電気毛のトリマーと動物の頭皮の毛をカット。麻酔中の乾燥からの保護のための動物の眼に眼軟膏の少量を適用します。 70％エタノールに続く10パーセントポビドンヨード溶液を、使用して頭皮を消毒する。 </li><li>安定した固定位置に頭部を保持する定位固定装置の上に動物をマウントします。手術中、私たちは低体温を防ぐために加熱パッドで動物を維持する。 </li><li>ラムダから目の間に伸びる、メスを用いて頭皮上正中前後切開を加えます。わきの皮膚を引っ張ると十分に左頭蓋骨を露出させる止血を使用してください。 </li><li>左頭蓋骨の長方形のカットを確認し、骨膜を掻きにメスを使用。このステップでは、出血を減らし、頭蓋骨にドリルを容易にするでしょう。 </li></ol> 3。アンダーカットを作る<ol><li>露出した頭蓋骨のエリアへの滅菌生理食塩水を少量追加し、その後、血液をきれいにし、エリアを乾燥させるいくつかの綿棒を使用してください。 </li><li>手術用顕微鏡下で、左側の頭蓋骨の中心（約左感覚運動皮質上記）の長方形の溝（ラットでは〜5 × 7 mmの、マウスの4 × 5 mm）を掘削開始。頭蓋骨に生理食塩水の滴を適用すると、掘削を容易にし、熱を放散する。掘削された骨の約2 / 3の深さの後に、過剰な生理食塩水を削除し、Q -ティップで掘削エリアを清掃してください。骨のセンターピースは、鉗子の優しいタッチ時に移動可能になるまで、ゆっくりと慎重に深くドリル。 </li><li>慎重に骨の縁に鉗子の鋭い先端を挿入し、ゆっくりと左半球を公開するために鉗子を持ち上げて、骨の中央部分を削除してください。 </li><li>手術用顕微鏡下で、針の先端とターゲット皮質領域の可視化を維持するために矢状方向に針と正中線への支持プレートに垂直、傾斜デバイス少し尾側アンダーカットデバイスをホールドし、向き。頭蓋窓の真ん中に、上矢状縫合の外側領域に1〜2ミリメートルに針の先端を目指し、大型船の直接侵入を避ける。 </li><li>血管を倹約するように硬膜を介してと軟膜下の水平方向に針を挿入します。アンダーカット装置の底部に座る頭蓋窓の両方のエッジ上に針を皮質表面（図1）に通常の配置されるように。手で頭蓋窓の両方のエッジでデバイスを置くと、大幅に、次の手順の実行中に手ぶれを軽減または排除されます。 PIAの下に針を持ち上げ、その後徐々に針が深く行くことができないまで、皮質間のカットを作成するために下げる。 °離れてVIは、アンダーカットの半円形の白質/深い層を作成するには、正中線から〜135を回転させる。その後、PIAの下に再び針を上げる。後方デバイスを傾け、針を撤回する。 </li><li>それに付随して、上記の手順（ステップ2.5）繰り返すが、ウィンドウの横方向の端で追加の皮質間のカットを作成するように針を回すことなくできます。これにより、より完全な皮質分離が作成されます。 </li><li>保護のための頭蓋窓の上にプラスチックフィルム（6 × 6 mm）の部分を置き、頭皮を縫合。完全に麻酔から回復するまで加熱されたパッドの上に動物を置きます。 </li></ol> 4。代表的な結果： コロナ皮質スライスは、アンダーカット手術の成功を確認するために調製することができる。 &gt; 2週間、手術後の準備のスライスでは、皮質間のとアンダーカットは、顕微鏡の低消費電力の目標（図2）の下に認められるです。部分的に分離された皮質は、通常わずかに薄くなり、てんかん様の活動は、フィールドの潜在的な記録（図3）と皮質スライスの大部分で検出することができます。 対照的に、白質のまたは深く皮質層に大きな穴、病変皮質の間伐劇的な、または上または下白質は、さらなる実験のための脳が使用できなくなるだろうという誤ったカットの形成。 <img src="/files/ftp_upload/2840/2840fig1.jpg" alt="図1"/> 図1。アンダーカット装置の構造と応用。導くチューブ（2）ステンレス鋼または透明プラスチックで作られて支持板（1）の上に接着される。注射針（3）先端まで〜3 mmの誘導管や曲がりが挿通されている。針は、プラスチックチューブ製の停止は、針の垂直方向の移動範囲が約1.2 mmとそれぞれP21マウスとラットでの使用のための〜1.5 mmに制限されているように、針の上端に接着される。小さな銅線のセグメントは、曲がった針の方向のための針の指標として、ハンドルの下に締結されている。カットが軟膜表面に平行に行われるようにデバイスを傾けると頭蓋窓のエッジの両方に接触していることに注意してください。 <img src="/files/ftp_upload/2840/2840fig2.jpg" alt="図2"/> 図2。アンダーカットスライスの代表画像。 P48ラットにおけるアンダーカットの病変の後二週間を作製したスライスのA.蛍光画像。カット傷は蛍光色素のDIIによって標識した。右側の白い矢印は、皮質間のカットを示しており、底部の矢印は、IV層と白質の間の境界も合格したことをアンダーカットを示している。 <img src="/files/ftp_upload/2840/2840fig3.jpg" alt="図3"/> 図3。アンダーカットの脳のスライスからアンダーカットスライス。フィールドの潜在的な録音からフィールドの潜在的な記録は、損傷した皮質組織の興奮を示唆し、てんかん様活性を示した。

<ol>
	<li>Hoffman, S. N., Salin, P. A., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neocortical+epileptogenesis+in+vitro.">Chronic neocortical epileptogenesis in vitro.</a> J Neurophysiol. 71, 1762-1773 (1994).</li><li>Topolnik, L., Steriade, M., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperexcitability+of+intact+neurons+underlies+acute+development+of+trauma-related+electrographic+seizures+in+cats+in+vivo.">Hyperexcitability of intact neurons underlies acute development of trauma-related electrographic seizures in cats in vivo.</a> Eur J Neurosci. 18, 486-496 (2003).</li><li>Graber, K., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Models of Seizures and Epilepsy. , 477-493 (2005).</li><li>Nita, D. A., Cisse, Y., Timofeev, I., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+propensity+to+seizures+after+chronic+cortical+deafferentation+in+vivo.">Increased propensity to seizures after chronic cortical deafferentation in vivo.</a> J Neurophysiol. 95, 902-913 (2006).</li><li>Sharpless, S. K., Halpern, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+excitability+of+chronically+isolated+cortex+studied+by+means+of+permanently+implanted+electrodes.">The electrical excitability of chronically isolated cortex studied by means of permanently implanted electrodes.</a> Electroencephalogr Clin Neurophysiol. 14, 244-255 (1962).</li><li>Echlin, F. A., Battista, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+Seizures+from+Chronic+Isolated+Cortex.">Epileptiform Seizures from Chronic Isolated Cortex.</a> Arch Neurol. 9, 154-170 (1963).</li><li>Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptogenic+neurons+and+circuits.">Epileptogenic neurons and circuits.</a> Adv Neurol. 79, 665-684 (1999).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neuropathology+and+impact+of+perinatal+brain+damage.+II:+white+matter+lesions+of+the+neocortex.">Developmental neuropathology and impact of perinatal brain damage. II: white matter lesions of the neocortex.</a> J Neuropathol Exp Neurol. 56, 219-235 (1997).</li><li>Jin, X., Prince, D. A., Huguenard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitatory+synaptic+connectivity+in+layer+v+pyramidal+neurons+of+chronically+injured+epileptogenic+neocortex+in+rats.">Enhanced excitatory synaptic connectivity in layer v pyramidal neurons of chronically injured epileptogenic neocortex in rats.</a> J Neurosci. 26, 4891-4900 (2006).</li><li>Li, H., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+in+chronically+injured,+epileptogenic+sensory-motor+neocortex.">Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex.</a> J Neurophysiol. 88, 2-12 (2002).</li><li>Salin, P., Tseng, G. F., Hoffman, S., Parada, I., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+sprouting+in+layer+V+pyramidal+neurons+of+chronically+injured+cerebral+cortex.">Axonal sprouting in layer V pyramidal neurons of chronically injured cerebral cortex.</a> J Neurosci. 15, 8234-8245 (1995).</li><li>Avramescu, S., Nita, D. A., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+post-traumatic+epileptogenesis+is+associated+with+loss+of+GABAergic+neurons.">Neocortical post-traumatic epileptogenesis is associated with loss of GABAergic neurons.</a> J Neurotrauma. 26, 799-812 (2009).</li><li>Avramescu, S., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+strength+modulation+after+cortical+trauma:+a+role+in+epileptogenesis.">Synaptic strength modulation after cortical trauma: a role in epileptogenesis.</a> J Neurosci. 28, 6760-6772 (2008).</li></ol>

利害の衝突は宣言されません。

アンダーカットモデルは、心的外傷後てんかんを研究するための非常に効率的なシステムです。典型的な手術が終了するのは約20〜30分かかり、誘発または自発的なてんかん様活動二週間手術1月2日後にほとんどの動物からのスライスに記録することができます。さらに重要なことは、このモデルでは、出血、炎症、浮腫、軸索切断や神経細胞死7などの外傷性脳損傷後の変化?...

<table border="1"><tbody><tr><td> 材料名 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> Foredomマイクロモータキット</td><td>機器の</td><td> Foredom </td><td> K.1070 </td></tr><tr><td> 1.5インチ22ゲージの注射針</td><td>材</td><td> BDの会社</td><td> 305156 </td></tr><tr><td> 1.5インチ25ゲージの注射針</td><td>材</td><td> BDの会社</td><td> 305127 </td></tr><tr><td>シアノアクリレート接着剤</td><td>材</td><td>テッドペラ</td><td> 14450 </td></tr></tbody></table>

preparing undercut model of posttraumatic epileptogenesis in rodents

部分的に分離された皮質が（&quot;アンダーカット&quot;）外傷後てんかんの動物モデルである。外科手術は大脳皮質の特定領域は、主に隣接する皮質と1-3皮質下の領域から分離されるように（アンダーカット）の白質の下に感覚運動皮質を切開して行います。手術後2週間以上の待ち時間の後に、てんかん様放電は、げっ歯類1からの脳切片に記録することができ、電気的または行動発作は、ネコやサル4-6と他の種からin vivoで観察することができます。この確立された動物モデルは、生成するために効率的ですし、外傷性脳損傷のいくつかの重要な特徴を模倣しています。しかし、それは技術的にフリーハンドを持つ小型の齧歯類の脳で正確な皮質病変を作るしようと挑戦している。最初にスタンフォード大学1でデビッドプリンスの研究室で確立された手順に基づいて、ここで我々はマウスおよびラットにおけるこのモデルの準備のための手術を実行するための改良手法を提案。我々は単純な手術装置を作ると、より正確で一貫性のある結果を生成するために深さと角度を切るのよりよい制御を得るためにそれを使用する方法を示します。デバイスは、作るのは簡単です、と手順を学ぶために迅速です。この動物モデルの生成は、外傷後てんかん発生のメカニズムに関する研究のための効率的なシステムを提供します。

Watch this Scientific Journal Video about Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents at JoVE.com

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

部分的に分離された皮質が（&quot;アンダーカット&quot;）外傷後てんかんの効率的な動物モデルである。ここでは、新たな外科手術デバイスを作成し、このモデルを生成するために、より正確で一貫性のある病変を作ってそれを使用する方法を示します。

げっ歯類で心的外傷後てんかん原のアンダーカットモデルを準備

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

preparing-undercut-model-of-posttraumatic-epileptogenesis-in-rodents

Research

JoVE Journal

Neuroscience

9.3K ビュー.  Indiana University School of Medicine. 部分的に分離された皮質が（"アンダーカット"）外傷後てんかんの効率的な動物モデルである。ここでは、新たな外科手術デバイスを作成し、このモデルを生成するために、より正確で一貫性のある病変を作ってそれを使用する方法を示します。

ビデオ: Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

げっ歯類における音響驚愕の慣れとプレパルス抑制

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

神経科学

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

動作げっ歯類の可動シリコンプローブによるニューロンの大規模なレコーディング

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

げっ歯類における行動聴力検査のための低コストのセットアップ

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

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, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

げっ歯類での同時のfMRIと脳深部刺激

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

げっ歯類でのアクリル樹脂成形ベースヘッド固定法

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

げっ歯類での後根神経節の脊髄および単離の油圧押出

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

警告齧歯動物で経頭蓋脳の電気的刺激

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

齧歯動物の槽注入カニューレ

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

頭蓋内薬物療法と痛みの試金の齧歯動物

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...