We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).

倫理声明：動物を対象とする手順は筑波大学施設内動物実験委員会によって承認されています。 EEG / EMG記録のために電極とケーブルの作製<ol><li>以下の手順に従って、EEG / EMGの記録電極を準備します。 注記：電極は、使い捨てであり、1動物のためにのみ使用することができます。慎重に、すべてのコネクタの配線構成を計画します。正しい方向用のコネクタ上のマークを配置します。 <ol><li> 2センチのステンレス鋼線に4ピン・ヘッダの各端子を半田付けします。簡単に言えば、ピンにワイヤーの一端を保持するワイヤーピンジョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。ピンにあまりにも多くの熱を加えないように注意してください。それ以外の場合は、ピンの周りのプラスチックが溶けてしまいます。 </li><li>先頭にピンヘッダに接続されている2線のそれぞれの自由端をはんだ付けし1.0 mmの直径のステンレス鋼製ねじの。簡単に言えば、ねじ頭部下のスレッドに、ワイヤの自由端を保持するワイヤースクリュージョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。ネジなしの2線が筋電図記録電極として機能する一方、ネジで2ワイヤは、脳波記録電極として機能します。 </li><li> EMG信号の品質を高めるために、EMG電極の端で絶縁の1ミリメートルを取り除くためにハサミを使用してください。 </li><li>完全EEG / EMGの録音中に電気的なノイズを低減するために細い木の棒や歯のピックを使用してエポキシ系接着剤ですべてのはんだ付けのピンをカバーしています。 </li></ol></li><li>以下に説明するようにスリップリングに電極を接続するためのケーブルを用意します。このケーブルを再利用することができます。 <ol><li> 30 cmのフラットケーブルの線で4ピンFFC / FPCコネクタの各ピンを半田付けします。簡単に言えば、ホットはんだごてを配置し、ピンに電線の被覆をむいた部分を保持ワイヤーピンジョイントの上に、とだけで十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。 注：EEG / EMG記録のために使用される実験動物用ケージの高さに適したフラットケーブルの長さを選択してください。 </li><li>はんだは、フラットケーブルのもう一方の端のワイヤーの先端にソケットを圧着します。簡単に説明すると、ワイヤの被覆の自由端に圧着ソケットを保持するワイヤーソケットジョイントにホットはんだごてを置き、ちょうど十分なはんだが関節にスムーズに動作することを保証するために、いくつかのはんだを溶かします。 </li><li> 4位圧着ハウジングにそれぞれ圧着ソケットを挿入します。 </li><li>完全に細い木の棒や歯のピックを使用してエポキシ系接着剤で圧着ソケットをカバーしています。 </li></ol></li></ol>マウスヘッドの電極の2注入（所要時間：約20分） <ol><li>手術前にホットビーズ滅菌器内のすべての手術器具を滅菌します。男性のマウスを麻酔（10から20週齢、20〜30グラム）ペントバルビタール（50mg / kg）の腹腔内注射です。マウスが深くつま先をつまんで麻酔されていることを確認した後、バリカンで頭と首の毛を剃ります。 </li><li>定位フレームにマウスを移動し、2耳のバーの間の頭を固定します。乾燥を防ぐために、目にワセリンを適用します。アルコールで剃られ、肌を清潔にし、頭蓋骨を露出するようにメスで正中線に沿ってそれをカット。オープン手術領域を維持するために皮膚をクリップ。 </li><li>超硬カッター（ドリルサイズ：0.8 mmの直径）を使用して、頭蓋骨の中に、ドリル2穴を、前頭皮質領域（正中線に横1.5ミリメートルをブレグマの1mm前方）上の1つと頭頂領域の上に他の（ 1ミリメートルラムダの前方、正中線に横1.5ミリメートル）右半球の、Paxinosとフランクリンの定位座標に応じた。23 </li><li>宝石商のドライバーを使用することで、穴にステンレス製脳波記録ネジを配置し、2作る - 各SCRE 2.5ターン皮質の上に硬膜外位置決め用ワット 注意：脳組織への損傷を防止するにはあまりにも深いネジを挿入しないでください。ネジがしっかりと頭蓋骨に固定されていることを確認してください。これは、複数のレコーディング（典型的には、1ヶ月以上）の長い期間の間に安定したEEG信号を有することが重要です。ウィグリーネジは脳波アーティファクトを生成し、実験スケジュールの終了前にオフに来るかもしれません。 </li><li>電極アセンブリ修正歯科用セメントで頭蓋骨とカバーに瞬間接着剤で（参照：セクション1.1を、ピンが上向き）。僧帽筋（首）筋肉のピンセットで二国間の小さな穴を作り、穴にEMG電極として機能するステンレス鋼線を挿入します。筋肉の露出を避けるために絹糸（直径0.1mm）で皮膚を縫合。 </li><li>定位フレームからマウスを外します。細菌感染を避けるために、マウスを、アンピシリン（100mg / kg）および腹腔内メロキシカム（1ミリグラム/キログラム）を管理し、それぞれ、手術後の痛みを軽減します。柯熱パッドの上にマウスをEPと、それは胸骨横臥位を維持するのに十分な意識を取り戻したまでそれを監視します。ハウスは、個別に回復中のマウスは、他の動物によって電極の除去を回避し、手術後の最初の日に腹腔内、メロキシカム（1ミリグラム/キログラム）を管理します。 </li></ol> 3.記録と脳波/ EMGデータの取得<ol><li> 1週間の回復期間、住宅防音記録チャンバーに入れた実験ケージに個別に各マウスの後。 12時間の光/ 12時間暗のサイクルを制御し、自動的に23±1℃の周囲温度を維持し、（照度〜100ルクス、08:00に点灯）。 </li><li>記録ケーブルにマウスの頭の上にEEG / EMG電極アセンブリを接続します。記録ケーブルが（マウスの動きがケーブルのねじれによって制限されないように設計された）スリップリングおよびEEG / EMG信号増幅器に接続されていることを確認。フィルタEEG / EMG信号（脳波、0.5-64ヘルツ; EMG、16-64 Hz）は、アナログ-デジタル変換器（A / D）で128ヘルツのサンプリングレートでデジタル化し、最終的にはEEG / EMG記録ソフトウェアを実行するコンピュータ（表「材料」、4 番目のエントリに記録下から）。 </li><li>記録チャンバー内の3日間 - 2のためのマウスを慣らします。 EEG / EMG記録を腹腔内薬物投与が含まれる場合は、ゆっくり薬物投与時に各慣れ日にマウスを処理します。 </li><li>その後、EEG / EMG記録ソフトウェア（表「マテリアル」、下から4 番目のエントリ ）を起動します。 <ol><li> 「データファイル情報]タブを選択し、ファイル名の横にあるボックスをクリックします。ファイル名を入力し、[保存]をクリックします。 </li><li> 「記録条件」タブを選択し、記録されるすべてのEEG / EMGチャンネルを選択します。 </li><li> 「記録条件」タブで、サンプリング周波数（128 Hz）を選択します。 </li><li>選択したチャンネルが目に正しく表示されているかどうかを確認してくださいE &#39;チャンネル情報」タブをクリックします。 </li><li> 「タイマー設定」タブを選択し、脳波と筋電図を表示するには、「モニター」をクリックします。 </li><li> EEG / EMG信号が正しく表示されるか確認してください。 </li><li> 「タイマー設定」タブを選択し、「メインタイマー」領域での記録の開始と終了のクロック時刻を設定します。 </li><li>録音を開始するには「タイマー設定」タブの「モニター」をクリックします。 </li></ol></li><li>ベースラインの下に録音EEG / EMG信号（ すなわち 、自由に振る舞うマウスの睡眠/覚醒行動）と異なる処理条件（ 例えば 、カフェインの投与または偽の治療）、数日間。最後の実験日後にペントバルビタール（200mgの/ kg）の腹腔内注射でマウスを安楽死させます。 </li></ol> EEG / EMGデータに基づく行動状態の4得点<ol><li> EEG / EMG解析のためのソフトウェアを起動します（表「マテリアル」、下から4 番目のエントリ ）。手順3で製造したEEG / EMG生データ（.kcdファイル）を開き「スリープ」タブをクリックし、エポックの時間を選択します。 10秒を選択します。 </li><li> 「スリープ」タブをクリックし、自動的に3段階にすべての10秒エポックを採点する「マルチスクリーニング」を選択（ すなわち 、NREMおよびREM睡眠を、および覚醒）脳波と筋電図の振幅の基礎とパワースペクトル解析に脳波の。3 </li><li> 「脳波のためのFFT条件]をクリックします。 </li><li>パワースペクトル分析のためのパラメータを設定し、[256基準点（EEGの2秒に相当）、ハニング窓関数、及びエポック当たり5スペクトルの平均]。3「OK」をクリックします。 </li><li>自動スクリーニングを開始する「スクリーニング開始]をクリックします。得点データ（.rafファイル）を開きます。 </li><li>代表的な結果、 図1B および表1を参照してください （自動スクリーニングの結果を確認し、必要に応じて、標準的な基準に基づいて手動でそれらを修正</strong&gt;）。2、3簡単に説明すると、クリックして、間違って得点エポック上でマウスの左ボタンを押したまま、誤っ得点エポックの文字列全体にカーソルをドラッグします。マウスの左ボタンを離すと、ポップアップウィンドウに正しい行動の状態を選択します。 注：時々、2警戒状態間の遷移でのエポックは、一つの状態に明確に得点することは困難です。このような場合には、エポックは、表向きの状態にスコア付けされるべきであり、同じ基準は、データの再現性を保証するために、実験全体を通して同様の時期に適用されるべきです。 </li></ol>

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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=Arousal+effect+of+orexin+A+depends+on+activation+of+the+histaminergic+system.">Arousal effect of orexin A depends on activation of the histaminergic system.</a> Proc. Natl. Acad. Sci. USA. 98 (17), 9965-9970 (2001).</li><li>Xu, Q., 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=A+mouse+model+mimicking+human+first+night+effect+for+the+evaluation+of+hypnotics.">A mouse model mimicking human first night effect for the evaluation of hypnotics.</a> Pharmacol. Biochem. Behav. 116, 129-136 (2014).</li><li>Cho, S., 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=Marine+polyphenol+phlorotannins+promote+non-rapid+eye+movement+sleep+in+mice+via+the+benzodiazepine+site+of+the+GABAA+receptor.">Marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the GABAA receptor.</a> Psychopharmacol. 231 (14), 2825-2837 (2014).</li><li>Liu, Y. Y., 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=Piromelatine+exerts+antinociceptive+effect+via+melatonin,+opioid,+and+5HT1A+receptors+and+hypnotic+effect+via+melatonin+receptors+in+a+mouse+model+of+neuropathic+pain.">Piromelatine exerts antinociceptive effect via melatonin, opioid, and 5HT1A receptors and hypnotic effect via melatonin receptors in a mouse model of neuropathic pain.</a> Psychopharmacol. 231 (20), 3973-3985 (2014).</li><li>Qu, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipocalin-type+prostaglandin+D+synthase+produces+prostaglandin+D2+involved+in+regulation+of+physiological+sleep.">Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17949-17954 (2006).</li></ol>

The authors Yujiro Tauguchi and Sayaka Kohtoh are employees of Kissei Comtech Co., Ltd that develops SleepSign software for acquisition and automatic scoring of EEG/EMG data used in this article.

このプロトコルは、低ノイズ、費用対効果の高い、高スループットの条件で睡眠と覚醒の評価を可能にするEEG / EMGの記録のためのセットアップについて説明します。 EEG / EMG電極ヘッドアセンブリのサイズが小さいために、このシステムは、マウスへの薬物の微量注入、光遺伝学（光ファイバの注入）または同時カニューレ注入と組み合わせて含むイントラ脳実験のための他のインプラントと...

技術の進歩は、多くの場合、神経生物学的プロセスの理解の飛躍を沈殿させています。例えば、ヒト頭皮から記録された電位は、正弦波の形をとった、の周波数は、被験者の覚醒のレベルに直接関連していたことを1929年にハンス・ベルガーの発見は、睡眠 - 覚醒の理解の急速な進歩につながりました同様に動物およびヒトの両方で規制、。1この日にelectroencephlogram（EEG）、筋電図（EMG）と組み合わせて、 すなわち 。骨格筋によって生成され、電気的活動は、ほぼすべての実験的および臨床のデータ「バックボーン」を表しヒトを含む、動物を振る舞うにおける皮質ニューロンの活動と行動や生理機能を相関しようとする評価。最も基本的な睡眠研究室では、これらのEEG記録は、Dを取得し、前記ケーブルベースのシステム（ 図1）を用いて実行されますATAは、[ 例えば 、高速フーリエ変換を適用（FFT）アルゴリズム]パターンとスペクトル解析にオフラインで供される記録される対象の警戒状態を決定する。2、3睡眠は急速眼球運動（REM）で構成され、ノンレム（NREM）睡眠。 REM睡眠は、急速な低電圧EEG、ランダムな眼球運動、および筋無緊張、筋肉を効果的に麻痺している状態によって特徴付けられます。身体は、脳の大部分は切断され、深い眠りであるように見えるのに対し、脳活動は、覚醒のに似ているので、REM睡眠はまた、逆説睡眠としても知られています。対照的に、運動ニューロンは、NREM睡眠中に刺激されるが、何の眼球運動は存在しません。脳波の4 Hzの - 人間ノンレム睡眠は、ステージ4は深い眠りまたは徐波睡眠と呼ばれ、0.5の間のデルタ活性を有する大規模な、遅い脳波によって識別される、4段階に分けることができます。一方、ラットのAのような小さい動物でのノンレム睡眠の相間の細分化、NDマウスは、ヒトで見られるように、彼らは睡眠の長い連結期間を持っていない主な理由は、確立されていません。 長年の間、及びEEGの解釈に基づいて、睡眠 - 覚醒の調節のいくつかのモデルは、circuit-および体液性系の両方が提案されています。神経と睡眠の必要性の細胞の基礎あるいは、「スリープ・ドライブは、「未解決のままであるが、覚醒期間中に構築し、睡眠によって放散される恒常的圧力として概念化されています。一説には、内因性somnogenic要因は覚醒時に、それらの緩やかな蓄積が睡眠恒常的圧力の基盤であることを蓄積することです。液性因子によって調節されて眠る最初の正式な仮説が1892年に発表された4ローゼンバウムの仕事に入金されているが、それは石森5、6、独立ピエロン7で、100年以上前、睡眠促進化学物質の存在を実証しました。両研究者は催眠薬物質または「hypnotoxinsは「睡眠不足の犬の脳脊髄液（CSF）中に存在したことを、提案し、実際に証明した。8過去一世紀にわたり睡眠恒常性のプロセスに関与し、いくつかの追加の推定催眠薬の物質が同定されています（レビューについて、参考文献を参照してください。9）、プロスタグランジン（PG）は、D 2、10サイトカイン、11アデノシン、12アナンダミド、13およびウロテンシンIIペプチドを含む。14 初期および中期20 世紀にエコノモ15、16、Moruzziとマーグン17、およびその他による実験研究は、睡眠と覚醒の回線ベースの理論に影響を与えた結果を生成し、ある程度までの当時の体液理論を影睡眠。今日までに、いくつかの「回路モデル」は、それぞれ、（総説については、参考文献18を参照）の品質と量を変化させるデータによって通知、提案されています。一つのモデル例えば、徐波睡眠が前脳基底部におけるコリン作動性ニューロンからのアセチルコリン放出、主にブロカの対角バンドと物質のinominataの水平手足の核のconsisitingエリアのアデノシン媒介阻害を介して生成することが提案されています。 19睡眠/覚醒調節のもう一つの人気モデルは視床下部および脳幹における腹外側視索前野における睡眠導入ニューロンおよびウェイク誘導ニューロン間の相互抑制性の相互作用に基づいて、フリップフロップスイッチ機構について説明しています。18、20、21また、レム睡眠の中と外のスイッチングのために、同様の相互に抑制相互作用は脳幹内の領域のために提案されている、それは腹側中脳水道周囲灰白質、横橋被蓋、およびsublaterodorsal核である。22まとめると、これらのモデルは貴重な証明されていますヒューリスティックと睡眠研究の研究のために与えられた重要な解釈の枠組み。しかし、あなたがた睡眠 - 覚醒周期を調節する分子機構や回路のトンの完全な理解は、そのコンポーネントのより完全な知識が必要になります。以下に詳細ポリグラフ記録するためのシステムは、この目標を支援する必要があります。

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">4-pin header</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">A3B-4PA-2DSA(71)</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Ampicillin</td>
			<td style="width:143px;">Meiji Seika</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog-to-digital converter</td>
			<td style="width:143px;">Contec</td>
			<td style="width:167px;">AD16-16U(PCIEV)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Caffeine</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">C0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbide cutter</td>
			<td style="width:143px;">Minitor</td>
			<td style="width:167px;">B1055</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Crimp housing</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-4DS-2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crimp socket</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-30SC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dental cement (Toughron Rebase)</td>
			<td style="width:143px;">Miki Chemical Product</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epoxy adhesive</td>
			<td style="width:143px;">Konishi</td>
			<td style="width:167px;">#16351</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">FFC/FPC connector</td>
			<td style="width:143px;">Honda Tsushin Kogyo</td>
			<td style="width:167px;">FFC-10BMEP1(B)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flat cable</td>
			<td style="width:143px;">Hitachi Cable</td>
			<td style="width:167px;">20528-ST LF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Instant glue (Aron Alpha A)</td>
			<td style="width:143px;">Toagosei</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meloxicam</td>
			<td style="width:143px;">Boehringer Ingelheim</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital</td>
			<td style="width:143px;">Kyoritsu Seiyaku</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal amplifier</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sleep recording chamber</td>
			<td style="width:143px;">APL</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SleepSign software</td>
			<td style="width:143px;">Kissei Comtec</td>
			<td style="width:167px;">N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slip ring</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel screw</td>
			<td style="width:143px;">Yamazaki</td>
			<td style="width:167px;">N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel wire</td>
			<td style="width:143px;">Cooner Wire</td>
			<td style="width:167px;">AS633</td>
			<td></td>
		</tr>
	</tbody>
</table>

polygraphic recording procedure for measuring sleep in mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

図1Bは、異なる警戒状態でマウスの脳波の例を示します。 表1に示すように、脳波は4 Hz以下デルタリズムで大規模な、遅い脳波を示し、EMGが弱いまたは無信号を持っている場合、エポックは、ノンレム睡眠に分類されます。脳波は、6と10 Hzの間のシータ範囲で急激な低電圧脳波を示し、EMGは低振幅を示している場合エポックは、レム睡眠に分...

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

マウスで睡眠を測定するためのポリグラフ記録手順

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

polygraphic-recording-procedure-for-measuring-sleep-in-mice

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23.3K ビュー.  University of Tsukuba. The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

ビデオ: Polygraphic Recording Procedure for Measuring Sleep in Mice

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

マウスにおける運動協調性の測定

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

神経科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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, ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

後根潜在的記録によりマウスにおける脊髄シナプス前抑制を測定する<em&gt;インビボ</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

覚醒マウスにおける神経活性物質のMicroiontophoreticアプリケーションと組み合わせることで神経活動の細胞外記録

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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 ...

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 ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

マウスの二次運動皮質における二国間局場電位記録による半球横化の評価

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...