我々は、次の資金源を認める: NIH NINDS – K08NS101122 (JB), R01NS040337 (JK), R01NS044370 (JK), バージニア大学医学部 (JB).

すべての動物研究は、バージニア大学の施設動物のケアと使用委員会(IACUC)によって承認されました。
1. 電極建物/ケーブルビルディング
<ol><li>単極絶縁ステンレス鋼線(0.005"裸径0.008"被覆)を使用して、メスソケットコネクタ(メスレセプタクルコネクタ0.079)で接続された電極を作ります。</li>
	<li>特別なカスタムケーブルを使用して、動物をアンプに接続します。<ol>
<li>オスの4ピンコネクタ(オスコネクタ0.079")を4チャンネルユニティゲインインピーダンスマッチングオペアンプ(op-amp)に取り付けます。9 V バッテリに接続するワイヤに 10K の抵抗を接続します。オペアンプに接続されていないアース線は、バッテリーの中点として機能します。</li>
		<li>ケーブルの一方の端(AWG、0.012"OD)をop-ampに接続し、ケーブルのもう一方の端をアンプに接続します。</li>
	</ol>
</li>
</ol>2. 電極注入手術
<ol><li>下向きのフローフードで4-5%のイオブルランで子犬(出生後9日目)を麻酔します。手順の開始前に、ブピバカイン(0.02-0.05 mL、0.25%皮下局所浸潤)で子犬を注入する。</li>
	<li>動物が不動になったら、鼻コーンで立体的な段階に移します。耳をしっかり止めるには柔らかいので、イヤーバーの裏側を使用します。この年齢では、子犬は耳棒の尖った端を使用するために完全に発達した耳を持っていません。</li>
	<li>イオフルランの流れを下げ、2.5~3%に維持します。手術を通して子犬の安定した呼吸に目を光らせる。尾をつまんで痛みの反応を確認し、切開に進みます。</li>
	<li>頭蓋骨の切開領域をベタジンとアルコール(ヨウ素と70%エタノールの3サイクル交互)で殺菌する。切開領域が見えるように周囲の身体部分をドレープする。</li>
	<li>目の上から頭皮前部後部を開き、約0.5cmの皮膚を引き込みます。皮膚が頭蓋骨を露出して外側に引っ張るように、ステレオタックスステージ上でマウスの頭を再配置します。</li>
	<li>綿棒を使用して頭蓋骨に過酸化水素を塗布し、メスの刃を使用して頭蓋骨をきれいに削ります。頭蓋骨は非常に柔らかいです。擦り傷をしながら注意を払ってください。</li>
	<li>接着剤を1滴(約50μL)塗布し、そのアプリケータを使用して露出した頭蓋骨領域の周りに広げます。40sの紫外線に露出して接着剤をセットします。</li>
	<li>参照として、露出したブレグマを使用して座標を測定します。海馬のCA1領域に両側電極を移植する [-3.5 mm側側腹孔(DV),±2mm内側側面(ML)、-1.75mm深部(D)、頭頂皮質内の両側[-1.22mm DV、±0.5mm D]および小脳内の参照電極。32G針を使用して、マークされた領域に穴を開けます。</li>
	<li>頭蓋骨の表面から血液をきれいにします。立体的な腕の助けを借りて脳に女性のソケットコネクタに取り付けられた下の電極と歯科アクリルで所定の位置に固定します。脳に電極を埋め込む。ソケットコネクタヘッドセットは、歯科アクリルで接着頭蓋骨の上に座っています。</li>
	<li>電極が固定されたら、ケトプロフェン(5mg/kg)を皮間領域に皮下に注入します。子犬を母親と一緒に戻します。 注:一度に1つずつ導入するのではなく、一度にヘッドセットでゴミの半分を導入してください。これは、母親が子犬のヘッドセットを損傷するのを防ぎます。</li>
</ol>3. EEGのセットアップと記録(ベースライン/プレ傷害)
<ol><li>電極注入後24時間の回収後、各動物をEEG記録用の加熱(37°C)カスタムメイドのプレキシグラスチャンバーに入れます。このチャンバーは低酸素室としても機能します。</li>
	<li>柔軟なケーブル(カスタムメイドのオペアンプケーブル)を介してビデオ-EEG監視システムにチャンバー内の子犬を接続します。 メモ:ヘッドセットを設置すると、マウスは自由に移動可能で、動作に違いはありません。電極ワイヤーに取り付けられたら、ワイヤーは、子犬がチャンバー全体を自由に移動できるように、適切な量の緩みを提供するためにチャンバーテザー内で調整する必要があります。</li>
	<li>草のアンプを使用して1Kゲインで1000 HzでEEGデータをデジタル化します。ソフトウェア(例えば、LabChart Pro)を使用して、後でEEG信号(3〜70 Hzの間のバンドパスフィルタ)を確認してください。</li>
	<li>頸動脈結紮術のために動物を切断する前に、傷害前のベースラインEEGを30分間記録する。</li>
</ol>4. 左頸動脈結紮
<ol><li>下向きのフローフードで4-5%のイオブルランで子犬(出生後10日目)を麻酔し、ウォーターバスパッドに特別に配置されたセットアップに置きます。動物のスピージンを配置し、紙テープで前肢を固定します。<ol>
<li>イオブルランの流れを2~3%に下げる。痛みの反応のために尾をつまみ、手順全体の呼吸を監視します。</li>
	</ol>
</li>
	<li>ベタジンとアルコール(ヨウ素と70%エタノールの3サイクル)で首の左側の切開領域(下顎骨と鎖骨の間)を殺菌します。</li>
	<li>マイクロシザーを使用して、首の左側に約1cmの長さの切開を行います。解剖顕微鏡を用いて、皮下組織と皮膚を慎重に引き込み、頸動脈を露出させる。迷走神経(動脈に横向きに走る)を特定し、動脈から繊細に分離して引き込むのに注意してください。</li>
	<li>マイクロ鉗子を使用して動脈の下に長さ5cmの無菌シルク縫合糸を通す。動脈の周りに二重結び目の縫合糸を結んで、閉塞流れにします。</li>
	<li>余分な縫合糸をカットし、皮下組織と皮膚を引き戻すことによって露出した動脈を閉じます.切開を密封するために獣医の結合を使用してください。</li>
	<li>動物を温かいマットレスの上に置かれる室温の部屋の連続的なEEGの監視に戻す。チャンバーを開けないように、子犬のコア温度のスポット赤外線温度チェックを取ります。動物が低酸素症の前に1時間回復することを許可します。</li>
</ol>5. EEGと低酸素症
<ol><li>酸素モニターを介してチャンバー内のFiO2 (インスパイアされた酸素の一部)を継続的に監視します。</li>
	<li>100%N2 の60 L/minと100%O2のための0.415 L /分でチャンバーを洗い流す。チャンバーの酸素飽和度が12%に達したら、O2の流れを変えずにN2 の流れを10 L/分に減らします。小さな調整で、45分間8%でFiO2 を維持します。</li>
	<li>低酸素暴露の45分後、21%にFiO2 を戻す。</li>
	<li>子犬はチャンバーで回復し、2時間ポスト低酸素症のEEG上で監視しています。</li>
	<li>記録期間終了後、マウスをEEG記録から切り離し、母親に戻します。</li>
</ol>6. EEG分析
<ol><li>ラボチャートプロのビデオでEEGファイルを分析します。目の不自由な研究者に発作と背景パターンのEEGをマークしてもらいます17。発作は、高周波のリズミカルな鋭波放電(≥3xベースライン)を持つ10秒以上続く電写イベントと定義され、明確な進化17を有する。</li>
	<li>2番目の盲目の研究者レビューは、合意のためにランダムにマークされたイベントを持っています。</li>
	<li>マークされた各電気イベントの関連ビデオをレビューし、新生児げっ歯類行動発作スコア16に従って分析する。簡単に言えば、このスコアは0-6(不動から重度の強壮性クロニック挙動)の範囲です。発作セミオロジーをさらに特徴付けるには、横性(多焦点/二国間運動対焦点/一方的な混合)の行動を分析する。</li>
	<li>パワースペクトログラムを作成します。1024 データ ポイントのサイズを持つコサイン ベル データ ウィンドウを使用して高速フーリエ変換を使用します。87.5%のウィンドウオーバーラップの助けを借りてスペクトログラムに滑らかなX軸を作成します。電源をμV218として表現します。</li>
</ol>

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Childhood outcomes after hypothermia for neonatal encephalopathy.</a> New England Journal of Medicine. 366 (22), 2085-2092 (2012).</li><li>Pappas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+outcomes+after+neonatal+encephalopathy.">Cognitive outcomes after neonatal encephalopathy.</a> Pediatrics. 135 (3), 624-634 (2015).</li><li>van Schie, P. 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我々は、低酸素虚血発作の間に新生児マウスにおける連続的なビデオ-脳細胞モニタリングのためのモデルを提示した。EEGと連動したビデオ分析により、発作セミロジーの特徴付けが可能になります。EEGの分析は、パワースペクトログラムの抽出とバックグラウンド振幅分析を可能にします。
電極の配置中または不正確な配置時の損傷は結果に大きな影響を与える可能性?...

低酸素性虚血性脳症(HIE)は、新生児1000人に1.5人の新生児に影響を及ぼし、新生児発作の最も一般的な原因である1,2である。生き残った乳児は、脳性麻痺、知的障害、てんかん3,4,5などの様々な神経障害の危険にさらされています。
動物モデルは、低酸素虚血および新生児発作の病態生理を理解し、調査する上で重要な役割を果たす6,7。修飾されたヴァンヌッチモデルは、出生後10日目(p10)7,8に低酸素虚血(HI)を誘導するために使用される。この年齢のマウスの子犬は、人間の新生児の完全な用語に神経学的に大まかに変換します 9.
この傷害モデルと組み合わせて使用される連続ビデオ脳波(EEG)モニタリングは、新生児低酸素虚血性発作のさらなる理解と特徴付けを可能にする。これまでの研究では、ビデオ録画、限られたEEG記録、テレメトリEEG記録10、11、12、13、14、15、16など、げっ歯類の新生児発作を分析するための様々な方法を使用してきました。以下の原稿では、低酸素虚血中のマウスの子犬に連続したビデオEEGを記録する過程について詳しく述べる。新生児マウスの子犬における連続的なビデオEEGモニタリングのためのこの技術は、様々な疾患および発作モデルに適用することができる。

<table>
	<tbody>
		<tr>
			<td>SURGERY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ball Point Applicator</td>
			<td>Metrex Research</td>
			<td>8300-F</td>
			<td>i-bond applicator</td>
		</tr>
		<tr>
			<td>Cranioplast (Powder/Resin)</td>
			<td>Coltene</td>
			<td>H00383</td>
			<td>Perm Reline/Power</td>
		</tr>
		<tr>
			<td>I-Bond</td>
			<td>Kulzer GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LOOK Silk Suture</td>
			<td>Surgical Specialities Corporation</td>
			<td>SP115</td>
			<td>LOOK SP115 Black Braided Silk Non absorbable surgical suture</td>
		</tr>
		<tr>
			<td>RS-5168 Botvin Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5168</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>RS-5138 Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5138</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>UV light for I-Bond</td>
			<td>Blast Lite By First Media</td>
			<td>BL778</td>
			<td>UV ligth for I-bond</td>
		</tr>
		<tr>
			<td>Vannas Microdissecting Scissor</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5618</td>
			<td>Scissor for ligation</td>
		</tr>
		<tr>
			<td>Vet Bond</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>Vet Glue</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HYPOXIA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxidial</td>
			<td>Starr Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen sensor</td>
			<td>Medical Products</td>
			<td></td>
			<td>MiniOxI- oxygen analyzer/sensor for hypoxia rig</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Grass Amplifier</td>
			<td>Natus Neurology Incorporated</td>
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			<td>LabChart Pro</td>
			<td>ADI Instruments</td>
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			<td>Male Socket Connector 0.079&#34;</td>
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			<td>Texas Instruments, Dallas, TX, USA</td>
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</table>

continuous video electroencephalogram during hypoxia-ischemia in neonatal mice

低酸素虚血は新生児発作の最も一般的な原因である。動物モデルは、新生児発作や低酸素虚血の根底にあるメカニズムと生理学を理解するために重要です。本稿では、新生児マウスにおける連続的な脳波(脳波)モニタリングを行い、低酸素虚血中の脳波背景を解析する方法を説明する。ビデオとEEGを併用すると、発作のセミオロジーの記述と発作の確認が可能になります。この方法は、実験期間におけるパワースペクトログラムおよびEEG背景パターンの傾向を分析することも可能である。この低酸素虚血モデルでは、この方法は、損傷前のEEG記録が規範的ベースラインを得ることを可能にし、傷害および回復の間に。総モニタリング時間は、4時間以上母親から子犬を分離できないことによって制限されます。この原稿では低酸素虚血性発作のモデルを用いてきたが、新生児ビデオ脳Gモニタリングのこの方法は、げっ歯類の多様な疾患および発作モデルに適用することができる。

発作セミロジー
新生児低酸素-虚血曝露は、マウスの全身発作と焦点発作の両方をもたらす(図1A-C)。ビデオ脳波録画により、電解調査結果をビデオの動作と相関させます。これらの行動は、以前に公表された新生児げっ歯類行動発作スコア(BSS)16を用いて採点された。BSS に加えて、行動が焦点/一方的、二国間、?...

Watch this Scientific Journal Video about Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice at JoVE.com

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

本稿では、低酸素虚血を起こす新生児マウスにおける複数の深度電極を用いた連続的なビデオEEG記録の方法を説明する。

新生児マウスにおける低酸素虚血期における連続映像脳波

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

continuous-video-electroencephalogram-during-hypoxia-ischemia

Research

JoVE Journal

Neuroscience

3.3K ビュー.  University of Virginia. 本稿では、低酸素虚血を起こす新生児マウスにおける複数の深度電極を用いた連続的なビデオEEG記録の方法を説明する。

ビデオ: Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

マウスのビデオ眼球運動記録法

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

神経科学

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

マウスにおける胃腸の運動を分析するためにビデオ画像と時空間マップ

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

新生児マウスからの脊髄ニューロンの単離と培養

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

マウスにおける睡眠/覚醒状態のモニタリング中の神経回路の光遺伝学的操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

ヘモグロビンの脳室内注射による新生児脳室内出血のモデリング

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

<meta charset="utf8"></head><body>マウス感覚皮質の発達における神経活動マッピング

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>著者スポットライト:2光子顕微鏡と単一ニューロンイメージングによる神経回路形成の解読

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

新生児マウス感覚皮質における自発的活動の単一ニューロン分解能でのin vivo可視化

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...