この作業は、5R01NS051305-14および5R01NS076980-08をPJSに付与するNIHによって支援されました。

1. 実験準備
<ol><li>彼らは男性と女性のハエを分離し、バイアルに閉じるようにハエを収集します。 注:睡眠実験は、一般的に雌のハエで行われます。処女女性を収集することが重要です。嵌合雌は、データの分析を複雑にする幼虫に孵化する卵を産む。</li>
	<li><20のグループで単一のセックスの家のハエ。 注:社会的に豊かな環境(>50のグループ)での住宅ハエは、睡眠ドライブ6、13リバウンド睡眠の潜在的に交わる測定値を調節します。さらに、社会的な豊かさに続いて、睡眠は数日で減少します 6.したがって、ベースライン睡眠は、リバウンド睡眠の分析を複雑にする安定ではない。<20のグループでハエを維持することは、この潜在的な混乱を回避します。</li>
	<li>光と湿度制御された環境で3〜5日間バイアルにハエを入れておいてください。 注意:ハエの年齢と成熟度は睡眠に強く影響を与えます。睡眠は1日齢のハエで高く、4歳の3〜5日で安定します。ハエは通常12時間の光で維持されます:湿度50%で12時間の暗いスケジュール。</li>
</ol>2. 睡眠記録用チューブの準備
メモ:睡眠は運動活動モニターを使用して監視されます。モニターは5つのmmの直径の管の個々に収容される32のハエを収容できる。典型的には、遺伝子型は16または32ハエのグループで分析される。
<ol><li>一方の端にフライフードでチューブの適切な数を準備します。 注意:食事と代謝は睡眠33、34に影響することが知られているので、彼らが飼育されたのと同じ食べ物にハエを置くことを特に重要です。</li>
	<li>チューブの端をワックスで密封します。 注:睡眠不足とリバウンドは5日間の実験であり、適切に密封しないと食べ物が乾燥する可能性があります。適切に密閉されたチューブでは、食品を10日以上維持することができる。したがって、チューブの端部がうまく密閉されていることを確認することが重要です。しかし、ハエは濡れた食べ物にこだわることもあります。したがって、実験開始の1〜2日前にチューブを作るのに役立ちます。</li>
	<li>個別にウェイクを配置し、アスピレーターを使用して睡眠記録のための65ミリメートルの長いガラス管にハエを振る舞い、泡ストッパーでチューブの端を差し込みます。 注:ハエを睡眠記録のためにチューブに入れると、ハエはCO2 麻酔に再び曝されることはありません。吸引器は、一端をチーズクロスで覆ったゴムチューブから作られ、1 mLピペットチップに挿入されます。</li>
</ol>3. 睡眠の記録
<ol><li>睡眠を監視するために活動モニターにチューブでハエをロードします。 注意:スナップ岩は-60°から+60°から~10 sごとに前後に監視します。モニターは~5.9sの-60°で保持されています。モニターを保持しているトレイが-60°から+60°に移動し、〜1 sが+60°から-60°に戻るには〜2.9 sかかります。サイクル長は、モータに供給される電圧を調整することで、必要に応じて変更できます。<ol>
<li>チューブが正しい向きのアクティビティモニタに配置されていることを確認するように注意してください。正しい向きで、食べ物とチューブの端は、ハエが食品に押し込まないようにスナップの上部にあります。また、食べ物との終わりは睡眠記録のジャッキとモニターの側にある。これにより、アクティビティモニタをSNAPで正しく向け、同時に活動を監視しながら効率的な睡眠不足を実現できます。</li>
	</ol>
</li>
	<li>活動モニターを記録室に設置し、睡眠を監視します。</li>
	<li>ベースラインスリープを推定するために、少なくとも2日間の睡眠を監視します。 注:ハエがアクティビティモニターにロードされる日は、通常、ハエがチューブに収容されることに適応できるように適応日として除外されます。ベースライン睡眠は、ハエがロードされた日の翌朝から始まる少なくとも2日間(48時間)記録されます。</li>
	<li>アクティビティ記録ソフトウェア(例えば、午前8時から午前8時まで)を使用して、特定の日のライトの時間から前日のライトまでの1分のビンのハエの運動活動数を保存します。</li>
	<li>睡眠の発作のしきい値として5分の非活動を使用してカスタムマクロを使用して運動活動データから睡眠を推定する35. 注: 睡眠メトリックの数は、運動活動カウントから計算されます。これらは、24時間以上のmin /hで睡眠、24時間の合計睡眠時間、平均および最大昼時間と夜間睡眠の長さ36が含まれます。</li>
</ol>4. 睡眠不足と回復
<ol><li>ハエは可変長の時間(例えば、12時間、24時間および36時間)のために睡眠を奪われ得、回復睡眠はまた、様々な間隔(例えば、6時間、12時間、24時間および48時間)で評価することができ、実験的必要性によって回復の持続時間を決定する。睡眠回復は、睡眠の利得/損失プロットを使用して、または所定の間隔(例えば、6時間)にわたって回復した睡眠率を調べることによって視覚化することができます。</li>
	<li>2つのベースライン日にわたって睡眠が安定している場合は、3日目に、活動モニターをSNAPに入れ、一晩の睡眠不足を引き起こします。 注:ハエは睡眠時間8、32、37、38の範囲にわたって堅牢な睡眠リバウンドを示しますが、睡眠は確実にリバウンド睡眠を評価するために安定している必要があります。ベースライン日数間の睡眠の差が100分±場合、睡眠は安定している。</li>
	<li>モニタホルダーピン、モニタコードを接続し、背面にフードを付けて最後に正しく向き、前面にプラスチックバリアを配置して、アクティビティモニタが所定の位置に固定されていることを確認します(図1)。 注:スナップは、カムが10 s(図1)に1回回転するように設計されています。プラスチック製のインサートは、装置が「上」の位置にあるときにチューブを押し戻すことによってチューブをリセットします。チューブをリセットすることは、すべてのチューブが各サイクルの開始時に全範囲の運動を持つことを保証するために重要です。</li>
	<li>アクティビティモニタを抜き、一晩の睡眠不足の後のライトの上にすぐにSNAPからモニターを取り出します。 注:睡眠不足を終了し、ハエは一晩の睡眠不足の12時間後のライトの直後に回復に置かれることが重要です。ハエを回復に入れるのに20〜30分の遅延でさえ、リバウンド睡眠の範囲を妨げる可能性があります。</li>
	<li>ハエを記録室に置き、回復睡眠を監視するために2日間(48時間)邪魔されません。 注:記録室が他の実験に使用されている場合は、ハエの回復を刺激しないように細心の注意を払う必要があります。</li>
	<li>睡眠不足の量を計算します。個々のフライごとに、睡眠不足時に得られた睡眠とベースライン中の対応する時間の間の毎時差を計算します。時間ごとの差を合計して、失われた合計睡眠を計算します。</li>
	<li>回復した睡眠の量を計算します。個々のフライごとに、回復中に得られた睡眠とベースライン中の対応する時間の時間ごとの差を計算します。得られた合計睡眠を計算するために、毎時の差を合計します。 注:ハエが実際に睡眠不足であるかどうかは経験的です。したがって、実験者は睡眠不足率を調べる必要があります。フライが十分な睡眠量を失っていない場合は、分析から除外することができます。これは他の睡眠不足のアプローチに必要な場合がありますが、SNAPに必要なことはほとんどありません。より一般的には、睡眠不足が開始される前に、所定のハエで睡眠が安定しない場合があります。睡眠が安定していない場合、恒常性は計算できません。睡眠不足の開始前 ±に計算された睡眠の最大100分の最大差を包含の候補として受け入れます。時折、個々のハエの睡眠は24時間の日に不均一に分配されます(例えば、一部の個人は日中に睡眠クォータの60〜70%を得ることができ、夜間に12時間奪われたときに24時間の睡眠クォータのごく一部しか失わないかもしれません)。これらのハエは別々に評価することができます。</li>
	<li>各遺伝子型の回復期間の12時間、24時間、48時間にわたって(ベースラインに対して)回復された睡眠の平均割合を計算します。</li>
	<li>睡眠データから、ベースラインの平均および最大の昼間の睡眠の長さと、各遺伝子型の回復日数を計算します。 注:ハエのリバウンド睡眠は、睡眠量の増加と回復日の睡眠深さの増加によって特徴付けられる。睡眠の深さの尺度として睡眠の統合が使用されます。覚醒閾値は、睡眠深さの尺度としても使用できます。</li>
</ol>

<ol>
	<li>Nath, R. D., 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=The+Jellyfish+Cassiopea+Exhibits+a+Sleep-like+State.">The Jellyfish Cassiopea Exhibits a Sleep-like State.</a> Current Biology. 27 (19), 2984-2990 (2017).</li><li>Vorster, A. P., Krishnan, H. C., Cirelli, C., Lyons, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+sleep+in+Aplysia+californica.">Characterization of sleep in Aplysia californica.</a> Sleep. 37 (9), 1453-1463 (2014).</li><li>Zhdanova, I. V., Wang, S. Y., Leclair, O. U., Danilova, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+promotes+sleep-like+state+in+zebrafish.">Melatonin promotes sleep-like state in zebrafish.</a> Brain Research. 903 (1-2), 263-268 (2001).</li><li>Shaw, P. J., Cirelli, C., Greenspan, R. J., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlates+of+sleep+and+waking+in+Drosophila+melanogaster.">Correlates of sleep and waking in Drosophila melanogaster.</a> Science. 287 (5459), 1834-1837 (2000).</li><li>Hendricks, J. C., 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=Rest+in+Drosophila+is+a+sleep-like+state.">Rest in Drosophila is a sleep-like state.</a> Neuron. 25 (1), 129-138 (2000).</li><li>Ganguly-Fitzgerald, I., Donlea, J., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waking+experience+affects+sleep+need+in+Drosophila.">Waking experience affects sleep need in Drosophila.</a> Science. 313 (5794), 1775-1781 (2006).</li><li>Seugnet, L., Suzuki, Y., Vine, L., Gottschalk, L., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1+receptor+activation+in+the+mushroom+bodies+rescues+sleep-loss-induced+learning+impairments+in+Drosophila.">D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila.</a> Current Biology. 18 (15), 1110-1117 (2008).</li><li>Donlea, J. M., Thimgan, M. S., Suzuki, Y., Gottschalk, L., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+sleep+by+remote+control+facilitates+memory+consolidation+in+Drosophila.">Inducing sleep by remote control facilitates memory consolidation in Drosophila.</a> Science. 332 (6037), 1571-1576 (2011).</li><li>Seidner, G., 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=Identification+of+Neurons+with+a+Privileged+Role+in+Sleep+Homeostasis+in+Drosophila+melanogaster.">Identification of Neurons with a Privileged Role in Sleep Homeostasis in Drosophila melanogaster.</a> Current Biology. 25 (22), 2928-2938 (2015).</li><li>Li, X., Yu, F., Guo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+deprivation+specifically+impairs+short-term+olfactory+memory+in+Drosophila.">Sleep deprivation specifically impairs short-term olfactory memory in Drosophila.</a> Sleep. 32 (11), 1417-1424 (2009).</li><li>Melnattur, K., 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+conserved+role+for+sleep+in+supporting+Spatial+Learning+in+Drosophila.">A conserved role for sleep in supporting Spatial Learning in Drosophila.</a> Sleep. , 197 (2020).</li><li>Seugnet, L., Suzuki, Y., Donlea, J. M., Gottschalk, L., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+deprivation+during+early-adult+development+results+in+long-lasting+learning+deficits+in+adult+Drosophila.">Sleep deprivation during early-adult development results in long-lasting learning deficits in adult Drosophila.</a> Sleep. 34 (2), 137-146 (2011).</li><li>Donlea, J. M., Ramanan, N., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use-dependent+plasticity+in+clock+neurons+regulates+sleep+need+in+Drosophila.">Use-dependent plasticity in clock neurons regulates sleep need in Drosophila.</a> Science. 324 (5923), 105-108 (2009).</li><li>Bushey, D., Tononi, G., Cirelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+and+synaptic+homeostasis:+structural+evidence+in+Drosophila.">Sleep and synaptic homeostasis: structural evidence in Drosophila.</a> Science. 332 (6037), 1576-1581 (2011).</li><li>Huang, S., Piao, C., Beuschel, C. B., G&#246;tz, T., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+Active+Zone+Plasticity+Encodes+Sleep+Need+in+Drosophila.">Presynaptic Active Zone Plasticity Encodes Sleep Need in Drosophila.</a> Current Biology. 30 (6), 1077-1091 (2020).</li><li>Kirszenblat, 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=Sleep+regulates+visual+selective+attention+in+Drosophila.">Sleep regulates visual selective attention in Drosophila.</a> Journal of Experimental Biology. 221, (2018).</li><li>Singh, P., Donlea, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+Regulation+of+Sleep+and+Synapse+Pruning+after+Neural+Injury.">Bidirectional Regulation of Sleep and Synapse Pruning after Neural Injury.</a> Current Biology. 30 (6), 1063-1076 (2020).</li><li>Stanhope, B. A., Jaggard, J. B., Gratton, M., Brown, E. B., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+Regulates+Glial+Plasticity+and+Expression+of+the+Engulfment+Receptor+Draper+Following+Neural+Injury.">Sleep Regulates Glial Plasticity and Expression of the Engulfment Receptor Draper Following Neural Injury.</a> Current Biology. 30 (6), 1092-1101 (2020).</li><li>Kayser, M. S., Yue, Z., Sehgal, 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+critical+period+of+sleep+for+development+of+courtship+circuitry+and+behavior+in+Drosophila.">A critical period of sleep for development of courtship circuitry and behavior in Drosophila.</a> Science. 344 (6181), 269-274 (2014).</li><li>Kayser, M. S., Mainwaring, B., Yue, Z., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+deprivation+suppresses+aggression+in+Drosophila.">Sleep deprivation suppresses aggression in Drosophila.</a> Elife. 4, 07643 (2015).</li><li>Szuperak, 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=A+sleep+state+in+Drosophila+larvae+required+for+neural+stem+cell+proliferation.">A sleep state in Drosophila larvae required for neural stem cell proliferation.</a> Elife. 7, (2018).</li><li>Vaccaro, 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=Sleep+Loss+Can+Cause+Death+through+Accumulation+of+Reactive+Oxygen+Species+in+the+Gut.">Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut.</a> Cell. 181 (6), 1307-1328 (2020).</li><li>Kempf, A., Song, S. M., Talbot, C. B., Miesenb&#246;ck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potassium+channel+&#946;-subunit+couples+mitochondrial+electron+transport+to+sleep.">A potassium channel &#946;-subunit couples mitochondrial electron transport to sleep.</a> Nature. 568 (7751), 230-234 (2019).</li><li>Donlea, J. M., Pimentel, D., Miesenb&#246;ck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+machinery+of+sleep+homeostasis+in+Drosophila.">Neuronal machinery of sleep homeostasis in Drosophila.</a> Neuron. 81 (4), 860-872 (2014).</li><li>Liu, S., Liu, Q., Tabuchi, M., Wu, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+Drive+Is+Encoded+by+Neural+Plastic+Changes+in+a+Dedicated+Circuit.">Sleep Drive Is Encoded by Neural Plastic Changes in a Dedicated Circuit.</a> Cell. 165 (6), 1347-1360 (2016).</li><li>Pimentel, D., 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=Operation+of+a+homeostatic+sleep+switch.">Operation of a homeostatic sleep switch.</a> Nature. 536 (7616), 333-337 (2016).</li><li>Sitaraman, D., 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=Propagation+of+Homeostatic+Sleep+Signals+by+Segregated+Synaptic+Microcircuits+of+the+Drosophila+Mushroom+Body.">Propagation of Homeostatic Sleep Signals by Segregated Synaptic Microcircuits of the Drosophila Mushroom Body.</a> Current Biology. 25 (22), 2915-2927 (2015).</li><li>Foltenyi, K., Greenspan, R. J., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+EGFR+and+ERK+by+rhomboid+signaling+regulates+the+consolidation+and+maintenance+of+sleep+in+Drosophila.">Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila.</a> Nature Neuroscience. 10 (9), 1160-1167 (2007).</li><li>Seugnet, 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=Notch+signaling+modulates+sleep+homeostasis+and+learning+after+sleep+deprivation+in+Drosophila.">Notch signaling modulates sleep homeostasis and learning after sleep deprivation in Drosophila.</a> Current Biology. 21 (10), 835-840 (2011).</li><li>Seugnet, L., Galvin, J. E., Suzuki, Y., Gottschalk, L., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+short-term+memory+defects+following+sleep+deprivation+in+a+Drosophila+model+of+Parkinson+disease.">Persistent short-term memory defects following sleep deprivation in a Drosophila model of Parkinson disease.</a> Sleep. 32 (8), 984-992 (2009).</li><li>Tabuchi, 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=Sleep+interacts+with+a&#946;+to+modulate+intrinsic+neuronal+excitability.">Sleep interacts with a&#946; to modulate intrinsic neuronal excitability.</a> Current Biology. 25 (6), 702-712 (2015).</li><li>Melnattur, K., Zhang, B., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+flight+increases+sleep+and+identifies+a+novel+sleep-promoting+pathway+in+Drosophila.">Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila.</a> Science Advances. 6 (19), 2166 (2020).</li><li>Thimgan, M. S., Suzuki, Y., Seugnet, L., Gottschalk, L., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perilipin+homologue,+lipid+storage+droplet+2,+regulates+sleep+homeostasis+and+prevents+learning+impairments+following+sleep+loss.">The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and prevents learning impairments following sleep loss.</a> PLOS Biology. 8 (8), (2010).</li><li>Keene, A. C., 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=Clock+and+cycle+limit+starvation-induced+sleep+loss+in+Drosophila.">Clock and cycle limit starvation-induced sleep loss in Drosophila.</a> Current Biology. 20 (13), 1209-1215 (2010).</li><li>Shaw, P. J., Tononi, G., Greenspan, R. J., Robinson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+response+genes+protect+against+lethal+effects+of+sleep+deprivation+in+Drosophila.">Stress response genes protect against lethal effects of sleep deprivation in Drosophila.</a> Nature. 417 (6886), 287-291 (2002).</li><li>Andretic, R., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+sleep+recordings+in+Drosophila:+moving+beyond+sleep+time.">Essentials of sleep recordings in Drosophila: moving beyond sleep time.</a> Methods Enzymol. 393, 759-772 (2005).</li><li>Seugnet, 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=Identifying+sleep+regulatory+genes+using+a+Drosophila+model+of+insomnia.">Identifying sleep regulatory genes using a Drosophila model of insomnia.</a> Journal of Neuroscience. 29 (22), 7148-7157 (2009).</li><li>Bushey, D., Huber, R., Tononi, G., Cirelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Hyperkinetic+mutants+have+reduced+sleep+and+impaired+memory.">Drosophila Hyperkinetic mutants have reduced sleep and impaired memory.</a> Journal of Neuroscience. 27 (20), 5384-5393 (2007).</li><li>Geissmann, 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=Ethoscopes:+An+open+platform+for+high-throughput+ethomics.">Ethoscopes: An open platform for high-throughput ethomics.</a> PLOS Biology. 15 (10), 2003026 (2017).</li><li>Faville, R., Kottler, B., Goodhill, G. J., Shaw, P. J., van Swinderen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+does+your+mutant+sleep?+Probing+arousal+to+better+understand+sleep+defects+in+Drosophila.">How deeply does your mutant sleep? Probing arousal to better understand sleep defects in Drosophila.</a> Scientific Reports. 5, 8454 (2015).</li><li>Huber, R., 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=Sleep+homeostasis+in+Drosophila+melanogaster.">Sleep homeostasis in Drosophila melanogaster.</a> Sleep. 27 (4), 628-639 (2004).</li><li>Klose, M., Shaw, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-drive+reprograms+clock+neuronal+identity+through+CREB-binding+protein+induced+PDFR+expression.">Sleep-drive reprograms clock neuronal identity through CREB-binding protein induced PDFR expression.</a> bioRxiv. , (2019).</li><li>Dissel, 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=Sleep+restores+behavioral+plasticity+to+Drosophila+mutants.">Sleep restores behavioral plasticity to Drosophila mutants.</a> Current Biology. 25 (10), 1270-1281 (2015).</li><li>Gerstner, J. R., Vanderheyden, W. M., Shaw, P. J., Landry, C. F., Yin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty-acid+binding+proteins+modulate+sleep+and+enhance+long-term+memory+consolidation+in+Drosophila.">Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.</a> PLoS One. 6 (1), 15890 (2011).</li></ol>

著者らは開示するものは何もない。

ショウジョウバエの睡眠は、2000年に独立して特徴づけられ、2つのグループ4、5によって特徴付けられていた。これらの先駆的な研究では、ハエは穏やかな取り扱い(すなわち、手の剥奪)によって睡眠を奪われ、一晩の睡眠不足に対する堅牢なホメオスタティック応答を示すことが示された。重要なことに、睡眠不足の実験では、動物を目覚?...

睡眠は動物では普遍的に近いが、その機能は不明のままである。睡眠恒常性は、睡眠不足後の睡眠の代償的増加、睡眠の定義性の特性であり、多くの動物1、2、3、4、5の睡眠状態を特徴付けるために使用されてきた。
ハエの睡眠は、睡眠喪失4、5に対する堅牢な恒育応答を含む、人間の睡眠と多くの類似点を有する。ハエの睡眠に関する数多くの研究は、睡眠不足を使用して、延長覚覚から生じる有害な結果を調べることによって睡眠機能を推測し、睡眠の恒食調節を制御する神経生物学的メカニズムを決定することによって睡眠調節を理解してきた。したがって、睡眠不足のハエは、学習と記憶6、7、8、9、10、11、12、構造可塑性13、14、15、視覚注意16、神経損傷17、18、交配および攻撃的な行動19、交配および攻撃的な行動の障害を示すことが示された。20、細胞増殖21、及び酸化ストレス22に対する応答を、23に挙げ、いくつか挙げる。さらに、リバウンド睡眠を制御する神経生物学的メカニズムの調査は、睡眠ホメオスタット8、9、23、24、25、26、27、28、29を構成する神経機構に関する重要な洞察を生み出した。 .最後に、健康な動物の睡眠機能に関する基本的な洞察を明らかにすることに加えて、睡眠不足の研究はまた、病気の状態で睡眠機能に関する洞察を知らせた30、31。
睡眠不足は間違いなく強力なツールですが、睡眠不足実験では、動物を目覚めさせるために使用される刺激によって誘発されるものと、拡張覚醒から生じる表記型を区別することが重要です。手の剥奪または穏やかな取り扱いによる睡眠不足は、一般的に、最小限の破壊的な睡眠剥奪のための標準を設定すると考えられている。ここでは、睡眠無効装置(SNAP)を使用してハエを奪う睡眠プロトコルについて説明します。SNAPは、10年代ごとにハエに機械的刺激を与え、負のジオタキシスを誘発してハエを目覚めさせる装置です(図1)。SNAPは、高い睡眠駆動8、32を持つハエであっても、夜間の睡眠の>98%のハエを効率的に奪う。SNAPは強打敏感なハエで較正されており、SNAP内のハエの攪拌はハエに害を与えません。SNAPによる睡眠不足は、手の剥奪によって得られたものと同等のリバウンドを誘発する7.SNAPは、したがって、覚醒刺激の影響を制御しながら、ハエを奪う睡眠を取る堅牢な方法です。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Locomotor activity tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Tissue Prep Wax</td>
			<td>Thermo Fisher</td>
			<td>13404-122</td>
			<td>Wax used for sealing tubes</td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>Wale Apparatus</td>
			<td>244050</td>
			<td>We cut 5mm diameter Pyrex glass&#160;tubes into 65mm long tubes&#160;to record sleep. Pre-cut tubes&#160;can also be purchased.</td>
		</tr>
		<tr>
			<td>Nutri Fly Bloomington Formulation fly food</td>
			<td>Genesee Scientific</td>
			<td>66-113</td>
			<td>Labs might use their own fly food recipe. It is important that sleep be recorded on the same food that flies were reared in.</td>
		</tr>
		<tr>
			<td>Rotary glass cutting tool</td>
			<td>Dremel Multi Pro</td>
			<td>395</td>
			<td>Used to cut 65mm long glass tubes&#160;</td>
		</tr>
		<tr>
			<td>Monitoring Sleep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAM System and DAMFileScan software</td>
			<td>Trikinetics</td>
			<td></td>
			<td>Software used to acquire data from DAM monitors and save the acquired data in an appropriate format</td>
		</tr>
		<tr>
			<td>Data acquisition computer</td>
			<td>Lenovo</td>
			<td>Idea Centre AIO3</td>
			<td>A equivalent computer from any manufacturer can substitute</td>
		</tr>
		<tr>
			<td>Drosophila Activity Monitors</td>
			<td>Trikinetics</td>
			<td>DAM2</td>
			<td>These monitors are used to record flies&#39; locomotor activity</td>
		</tr>
		<tr>
			<td>Environment Monitor</td>
			<td>Trikinetics</td>
			<td>DEnM</td>
			<td>Not essential, but an easy way to monitor environmental conditions in the chamber where sleep is recorded</td>
		</tr>
		<tr>
			<td>Light Controller</td>
			<td>Trikinetics</td>
			<td>LC4</td>
			<td>A convenient way to control the timing of when the SNAP is turned on and off</td>
		</tr>
		<tr>
			<td>Power Supply Interface Unit for DAM</td>
			<td>Trikinetics</td>
			<td>PSIU-9</td>
			<td>Required for data acquisition computers to record Trikinetics locomotor acitvity data</td>
		</tr>
		<tr>
			<td>RJ11 connector</td>
			<td>7001-64PC</td>
			<td>Multicomp</td>
			<td>DAM monitors accept RJ11 jacks</td>
		</tr>
		<tr>
			<td>Splitters</td>
			<td>Trikinetics</td>
			<td>SPLT5</td>
			<td>Used to connect upto 5 DAM monitors</td>
		</tr>
		<tr>
			<td>Telephone cable wire</td>
			<td>Radioshack</td>
			<td>278-367</td>
			<td>Phone cables to acquire data from DAM monitors</td>
		</tr>
		<tr>
			<td>Sleep Deprivation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Gw INSTEK</td>
			<td>GPS-30300</td>
			<td>Power supply for the SNAP</td>
		</tr>
		<tr>
			<td>Sleep Nullifying Apparatus</td>
			<td>Washington University School of Medicine machine shop</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the sleep nullifying apparatus: a highly efficient method of sleep depriving drosophila

睡眠恒常性は、睡眠喪失後に観察される睡眠の増加であり、動物界全体の睡眠を識別するために使用される定義基準の1つである。その結果、睡眠不足と睡眠制限は、睡眠機能に関する洞察を提供するために一般的に使用される強力なツールです。しかし、睡眠不足の実験は、剥奪刺激自体が生理学と行動の観察された変化の原因である可能性があるという点で本質的に問題です。したがって、成功した睡眠不足技術は、動物を目覚めさせ、理想的には、意図しない結果を多数誘発することなく、堅牢な睡眠リバウンドをもたらすべきである。ここでは、ショ ウジョウバエのメラノガスターの睡眠剥奪技術について説明します。睡眠の無効化装置(SNAP)は否定的なジオタキシスを誘発するために10年代ごとに刺激を与える。刺激は予測可能であるが、SNAPは効果的に高い睡眠駆動を持つハエでも夜間睡眠の>95%を防ぐ。重要なことに、その後の恒恒動応答は、手による剥奪を用いて達成された応答と非常によく似ている。刺激のタイミングと間隔は睡眠喪失を最小限に抑え、刺激が生理学や行動に及ぼす非特異的な影響を調べるために変更することができる。また、睡眠制限や覚醒閾値の評価にも使用できます。SNAPは、睡眠機能をよりよく理解するために使用できる強力な睡眠破壊技術です。

広州S(Cs)は野生型株として使用した。ハエは12時間の光で維持された:12時間暗いスケジュール、そして睡眠は一晩で12時間奪われた。ベースライン日(bs)、睡眠剥奪日(sd)、および2つの回復日(rec1およびrec2)の睡眠プロファイルの検査は、ハエがSNAPで効果的に睡眠を奪われ、文献4、5で観察された報告と一致して日中に睡眠?...

Watch this Scientific Journal Video about The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila at JoVE.com

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

睡眠不足は、睡眠機能と調節を調査するための強力なツールです。我々は、睡眠排便装置を使用して ショウジョウバエ を奪い、剥奪によって誘発されるリバウンド睡眠の程度を決定するプロトコルを記述する。

睡眠の無効化装置:ショウジョウバエを奪う睡眠の非常に効率的な方法

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal ...

the-sleep-nullifying-apparatus-highly-efficient-method-sleep

Research

JoVE Journal

Neuroscience

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

3.3K ビュー.  Washington University School of Medicine. 睡眠不足は、睡眠機能と調節を調査するための強力なツールです。我々は、睡眠排便装置を使用して ショウジョウバエ を奪い、剥奪によって誘発されるリバウンド睡眠の程度を決定するプロトコルを記述する。

ビデオ: The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

薬物の強化のための新規装置および方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

神経科学

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

に概日リズムと睡眠のパラメータを研究するために運動活性を測定するショウジョウバエ</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

トータルRNAの単離およびダウンストリーム次世代シーケンシングと遺伝子発現プロファイリングのための離散マウスの脳領域のマイクロダイセクションのための非レーザーキャプチャー顕微鏡のアプローチ

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同時脳波、リアルタイム乳酸濃度の測定と齧歯類大脳皮質ニューロン活動のOptogeneticマニピュレーション

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

スプラーグ - ドーリーラットのメソッド節状神経節の注入

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

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.

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 ...

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 ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

新生児における信頼性の高い視覚イベント関連電位を得る方法

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

幼児期の昼寝中の睡眠依存性記憶統合の基礎となる神経機構の測定

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

マウスにおけるコフィリンを標的とするアデノ関連ウイルスを用いて操作した大脳皮質領域の電気コルチコチコ

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...