这项工作得到了国家卫生研究院向PJS提供5R01NS051305-14和5R01NS076980-08的支持。

1. 实验准备
<ol><li>收集苍蝇，因为它们封闭成小瓶，分离雄性苍蝇和雌性苍蝇。 注:睡眠实验通常与雌性苍蝇一起进行。收集处女很重要。交配的雌性会产卵，孵化成幼虫，使数据分析复杂化。</li>
	<li>房子飞的单性别组<20。 注:住房飞行在社会丰富的环境中（组>50）调节睡眠驱动器6，13潜在的混淆测量反弹睡眠。此外，随着社会富裕，睡眠将在几天内下降6。因此，基线睡眠不稳定，使反弹睡眠分析复杂化。将苍蝇分成20<组，避免了这种潜在的混淆。</li>
	<li>在光线和湿度控制的环境中，将苍蝇关在小瓶中3-5天。 注:苍蝇的年龄和成熟度对睡眠有很强的影响。睡眠是高在一天老苍蝇和稳定3-5天4岁。苍蝇通常保持在 12 h 光下:12 小时黑暗时间表，湿度为 50%。</li>
</ol>2. 准备用于睡眠记录的管子
注意:睡眠使用运动器活动监视器进行监控。监视器可以容纳 32 只苍蝇，分别安置在直径为 5 毫米的管子中。通常，基因型被分析成16或32只苍蝇。
<ol><li>准备适当数量的管子，一端有飞食。 注意:饮食和新陈代谢已知影响睡眠33，34，因此，将苍蝇放在饲养的相同食物上尤为重要。</li>
	<li>用蜡密封管子的末端。 注:睡眠剥夺和反弹是一个为期五天的实验，如果不能正确密封，食物可能会干涸。在密封良好的管子中，食物可以维持10天或更年。因此，确保管子的末端密封良好至关重要。然而，苍蝇也会粘在潮湿的食物上。因此，它有助于在实验开始前1-2天制造管子。</li>
	<li>单独放置唤醒，行为飞入65毫米长的玻璃管，使用吸气器进行睡眠记录，并用泡沫塞塞塞管末端。 注意:苍蝇在将苍蝇放入管中进行睡眠记录时，绝不会再次暴露在 CO2 麻醉中。吸气器由橡胶管制成，一端覆盖着奶酪布，并插入 1 mL 移液器尖端。</li>
</ol>3. 记录睡眠
<ol><li>将苍蝇装入管子中，放入活动监视器中，以监测睡眠。 注:SNAP 岩石每 10s 监控一次从 -60° 到 +60° 的来回监控。监视器以 -60° 为 5.9° 举行;托盘将显示器从 -60° 移动到 +60°和 +1 从 +60° 移回 -60°需要 +2.9 s。通过调整提供给电机的电压，可以根据需要更改循环长度。<ol>
<li>注意确保管子以正确的方向放置在活动监视器中。在正确的方向上，带食物的管子末端位于 SNAP 的顶部，以确保苍蝇不会被推入食物中。此外，末端与食物是在显示器的一侧与睡眠记录插孔。这允许活动监视器在 SNAP 中正确定位，以有效剥夺睡眠，同时监控活动。</li>
	</ol>
</li>
	<li>将活动监视器放在录音室中以监控睡眠。</li>
	<li>监测睡眠至少两天，以估计基线睡眠。 注:将苍蝇装载到活动监视器中的天数通常被排除在适应日之外，以便苍蝇能够适应被安置在管子中。基线睡眠记录至少两天（48小时），从第二天早上开始，苍蝇被加载。</li>
	<li>使用活动记录软件（例如，上午 8 点至上午 8 点），将苍蝇的活动计数从某一天的灯光时间保存到前一天的灯箱中 1 分钟。</li>
	<li>估计睡眠从运动活动数据与自定义宏使用5分钟的不活动作为一轮睡眠35的阈值。 注:从运动器活动计数中计算出许多睡眠指标。其中包括睡眠在分钟/小时超过24小时，总睡眠时间在24小时，平均和最大白天和夜间睡眠回合长度36。</li>
</ol>4. 睡眠剥夺和恢复
<ol><li>由于苍蝇的睡眠时间长度可能不同（例如，12小时、24小时和36小时），因此恢复睡眠也可以按不同间隔（例如，6小时、12小时、24小时和48小时）进行评估，从而根据实验需要确定恢复时间。睡眠恢复可以使用睡眠增益/损失图或检查在预定间隔内恢复的睡眠百分比（例如，6 小时）进行可视化。</li>
	<li>如果睡眠在两个基准日内稳定，在第三天，将活动监视器放入 SNAP 中，以剥夺夜间睡眠。 注:苍蝇在8、32、37、38的睡眠时间范围内会表现出强劲的睡眠反弹，但睡眠必须稳定才能可靠地评估反弹睡眠。当基准天数之间的睡眠差异± 100 分钟时，睡眠是稳定的。</li>
	<li>确保活动监视器固定到位，并插入监视器支架销、监视器电源线，并正确定位显示器，末端有食物，前面有塑料屏障（图1）。 注:SNAP 的设计使凸轮每 10s 旋转一次（图 1）。塑料插入在仪器处于"向上"位置时将管子推回，从而重置管子。重置管子对于确保所有管子在每个周期的开始都有全方位的运动非常重要。</li>
	<li>拔下活动监视器，并在夜间睡眠不足后立即将监视器从 SNAP 中取出。 注:必须终止睡眠剥夺，苍蝇在夜间睡眠剥夺12小时后立即在灯光下恢复。即使延迟20-30分钟将苍蝇放入恢复期，也会干扰反弹睡眠的程度。</li>
	<li>将苍蝇放在录音室中，在那里它们将不受干扰两天（48小时）以监测恢复睡眠。 注意:如果录音室被用于其他实验，必须格外小心，以避免刺激恢复的苍蝇。</li>
	<li>计算睡眠损失量。对于每只苍蝇，计算睡眠剥夺期间获得的睡眠与基线期间的相应小时数之间的小时差:汇总每小时的差异，计算总睡眠损失。</li>
	<li>计算恢复的睡眠量。对于每只苍蝇，计算恢复期间获得的睡眠与基线期间的相应小时数之间的小时差:汇总每小时的差异，以计算获得的总睡眠量。 注:苍蝇是否真的睡眠不足是经验。因此，实验者应该检查睡眠损失的百分比。如果苍蝇没有失去足够的睡眠，它可以排除在分析之外。虽然其他睡眠剥夺方法可能需要这样做，但 SNAP 很少需要这种方法。更常见的情况是，在开始睡眠剥夺之前，在给定的苍蝇中睡眠可能不稳定。如果睡眠不稳定，则无法计算平衡。我们接受在开始睡眠剥夺之前计算的最大睡眠差异为± 100 分钟， 作为加入候选者。有时，个体苍蝇的睡眠在24小时白天分布不均（例如，有些人在白天可能获得60-70%的睡眠配额，因此在夜间睡眠配额被剥夺12小时时，只能失去24小时睡眠配额的一小部分）。这些苍蝇可以单独评估。</li>
	<li>计算每个基因型恢复期超过 12 小时、24 小时和 48 小时的平均睡眠恢复百分比（相对于基线）。</li>
	<li>从睡眠数据中，计算基线上的平均和最大白天睡眠回合长度，以及每个基因型的恢复天数。 注意:苍蝇的恢复睡眠的特点是睡眠量增加，恢复日睡眠深度增加。睡眠巩固用作睡眠深度的测量。唤醒阈值也可以用作睡眠深度的测量标准。</li>
</ol>

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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. 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作者没有什么可透露的。

2000年，德罗索菲拉的睡眠被两组4、5组独立地描述。在这些开创性的研究中，苍蝇被温柔的处理（即手部剥夺）剥夺了睡眠，并显示出对夜间睡眠剥夺的强烈家庭静止反应。重要的是，对于任何睡眠剥夺实验来说，控制用于保持动物清醒的方法的潜在混淆效应至关重要。手部剥夺研究为研究苍蝇平衡设定了基准，作为剥夺苍蝇睡眠的最小破坏性手段。SNAP有效地剥夺了苍蝇>98%的夜间睡眠，并重要的是诱导睡眠反弹，可与手剥夺4，7获得。
自从对苍蝇睡眠进行定义的基础研究以来，已经开发出许多方法来以高吞吐量的方式评估苍蝇的睡眠平衡，方法包括7、9、39、40、41。我们调查了250篇关于苍蝇睡眠的论文，发现其中46%的发表文章报告使用睡眠不足来评估睡眠调节或功能（表1）。多种不同的方法有效地诱导了苍蝇的睡眠反弹。有趣的是，在评估睡眠反弹的研究中，用于睡眠剥夺和睡眠反弹的协议有所不同。具体来说，睡眠剥夺的持续时间（表2）和评估反弹的持续时间（表1）差别很大，可能使用不同的协议获得的结果的比较复杂化。众所周知，苍蝇的睡眠反弹在睡眠剥夺5后会持续长达48小时。因此，我们认为，当在 48 小时恢复期内评估自体静止反弹时，最好对给定的睡眠操作对平衡的影响进行彻底描述。
需要注意的是，白天剥夺苍蝇的睡眠并不能持续增加睡眠动力4。因此，在开灯时启动 24 小时睡眠剥夺协议并持续到第二天，与从熄灯开始的 12 小时睡眠剥夺协议相比，不会进一步增强恢复睡眠。事实上，计算的睡眠反弹可能较低，因为它将包括非家庭调节的白天睡眠，除了夜间睡眠。然而，白天睡眠剥夺不会诱发家庭静止反弹的观察可以用来控制睡眠剥夺方法的潜在混淆效应。因此，在SNAP中，苍蝇睡眠不足，与白天7时接受类似刺激的苍蝇相比。
除了用于完全睡眠剥夺，通过改变刺激的频率，SNAP也可以用来长期限制和碎片睡眠7，42，从而模仿人类慢性睡眠损失的条件。此外，通过以增加频率的步骤提供刺激，SNAP 还可用于测量唤醒阈值8。因此，SNAP 是有效剥夺和限制苍蝇睡眠、评估宿主反应和测量其他睡眠特征的一种便利方法。
SNAP 可以安装在标准的实验室苍蝇孵化器中，但肯定会干扰孵化器中的苍蝇，而孵化器中不是实验的一部分。幸运的是，SNAP可以放置在一个孤立的位置睡觉剥夺苍蝇，而不会打扰其他正在进行的实验。由于恢复睡眠是脆弱的，应小心，以确保恢复睡眠发生在一个安静的位置。
补充睡眠剥夺的研究，基因和药理学工具已经开发，以提高睡眠在苍蝇8，43，44。因此，能够方便地双向调节睡眠，将使飞行睡眠研究能够继续深入了解睡眠调节和功能。

睡眠在动物中几乎是普遍的，但其功能仍然不清楚。睡眠平衡，睡眠剥夺后睡眠的补偿性增加，是睡眠的一个定义属性，它被用来描述睡眠状态在一些动物1，2，3，4，5。
苍蝇睡眠与人类睡眠有许多相似之处，包括对睡眠损失的强健的家常反应4，5。许多关于苍蝇睡眠的研究都利用睡眠剥夺来推断睡眠功能，通过检查长时间清醒产生的不良后果，以及通过确定控制睡眠的内在调节的神经生物学机制来理解睡眠调节。因此，睡眠不足的苍蝇在学习和记忆方面表现出障碍6，7，8，9 ，10，11，12， 结构可塑性13，14，15， 视觉注意力16， 神经元损伤恢复17，18， 交配和攻击行为19，20、细胞增殖21，对氧化应激的反应22、23仅举几例。此外，对控制反弹睡眠的神经生物学机制的研究对构成睡眠家测器8、9、23、24、25、26、27、28、29的神经元机制产生了批判性的见解.最后，除了揭示健康动物睡眠功能的基本见解外，睡眠剥夺研究还对疾病状态30、31的睡眠功能进行了深入的了解。
虽然睡眠剥夺不可否认是一个强大的工具，任何睡眠剥夺实验，重要的是区分表型的结果，从延长清醒，那些诱导的刺激用于保持动物清醒。通过手部剥夺或温和处理剥夺睡眠通常被视为为最小破坏性睡眠剥夺设定标准。在这里，我们描述了一个协议，睡眠剥夺苍蝇使用睡眠无效设备（SNAP）。SNAP 是一种设备，每 10s 向苍蝇提供机械刺激，通过诱导负地球轴来保持苍蝇清醒（图 1）。SNAP有效地剥夺了苍蝇>98%的夜间睡眠，即使在高睡眠驱动器8，32苍蝇。SNAP 已校准了对爆炸敏感苍蝇的撞击，SNAP 中的苍蝇搅拌不会伤害苍蝇;睡眠剥夺与SNAP诱导反弹可与手剥夺7获得。因此，SNAP是一种强大的睡眠剥夺苍蝇的方法，同时控制刺激的效果。

<table><tbody><tr><td>&lt;strong&gt;Locomotor activity tubes&lt;/strong&gt;</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&amp;nbsp;tubes into 65mm long tubes&amp;nbsp;to record sleep. Pre-cut tubes&amp;nbsp;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&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Monitoring Sleep&lt;/strong&gt;</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&#039; 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>&lt;strong&gt;Sleep Deprivation&lt;/strong&gt;</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

睡眠平衡是睡眠损失后观察到的睡眠增加，是用于识别整个动物王国睡眠的决定性标准之一。因此，睡眠剥夺和睡眠限制是强大的工具，通常用来提供对睡眠功能的洞察。然而，睡眠剥夺实验本质上是有问题的，因为剥夺刺激本身可能是观察到的生理和行为变化的原因。因此，成功的睡眠剥夺技术应该让动物保持清醒，理想情况下，可以导致强劲的睡眠反弹，同时不会产生大量的意外后果。在这里，我们描述了一个睡眠剥夺技术， 为果蝇黑色素加斯特。睡眠无效装置 （SNAP） 每 10s 实施一次刺激，以诱导负地球轴。虽然刺激是可预测的，SNAP有效地防止>95%的夜间睡眠，即使在高睡眠驱动器的苍蝇。重要的是，随后的静止反应与使用手部剥夺实现的反应非常相似。可以修改刺激的时间和间距，以尽量减少睡眠损失，从而检查刺激对生理和行为的非特定影响。SNAP 还可用于限制睡眠和评估唤醒阈值。SNAP 是一种强大的睡眠中断技术，可用于更好地了解睡眠功能。

广州S（Cs）被用作野生型菌株。苍蝇保持在12小时的光线下:12小时的黑暗时间表，睡眠不足12小时过夜。对Cs苍蝇在基线日（bs）、睡眠剥夺日（sd）和两个恢复日（rec1和rec2）（图2A）的睡眠状况的检查表明，在SNAP中，苍蝇实际上睡眠不足，并且白天的睡眠恢复与文献4、5中的观察报告一致。SNAP在保持苍蝇清醒方面的有效性也体现在苍蝇在睡眠剥夺期间表现出的高活动（300-350计数/小时）上（图2B）。事实上，监测苍蝇在睡眠剥夺期间的活动计数可以是剥夺协议有效性和/或间接测量睡眠驱动的有用晴雨表。当睡眠剥夺无效时，苍蝇在剥夺期间就没那么活跃了。在高睡眠驱动器下的苍蝇在每次刺激后迅速入睡，并没有穿过管子那么多35。装置的倾斜角度和下降的速度对于确保苍蝇在不伤害它们的情况下有效地保持清醒至关重要。每个实验室可以通过调整弹簧（图 1B）和/或凸轮的大小和形状（图 1C和图1D，右图）来优化角度和速度。
为了定量估计睡眠剥夺和恢复的有效性，在剥夺期间失去的睡眠，然后在恢复日恢复，计算为每只苍蝇（图2C）。重要的是，在剥夺日和基线日之间基线睡眠没有显著变化（见 图2C中的0-12小时），表明这些苍蝇的睡眠是稳定的。在这12小时内（例如，±100分钟）的睡眠差异很大，表明睡眠不稳定。SNAP有效地剥夺了苍蝇98%的夜间睡眠>。苍蝇在前12小时恢复了20%的睡眠，而且没有像之前报道的那样在夜间恢复额外的睡眠。然而，苍蝇第二天开始恢复睡眠，因此它们恢复了48小时以上睡眠的36%（图2D）。30 - 40% 恢复睡眠超过 48 小时是相当典型的野生类型的苍蝇睡眠不足使用 SNAP.
睡眠平衡的特点是睡眠时间延长和剥夺后恢复期间睡眠深度增加。白天睡眠巩固通常用作睡眠深度的读数。睡眠巩固可以评估为全天的平均睡眠时间（图2E）。但是，随着恢复过程中睡眠压力的消散，平均睡眠时间将随着一天的进展而减少。因此，它经常有助于检查在最大睡眠回合持续时间的变化，可以提供一个更敏感的指标（图2F）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>睡眠剥夺方法</td>
			<td>论文总数</td>
			<td>% 论文/技术</td>
			<td>评估的 Avg 恢复</td>
		</tr>
<tr>
<td>折</td>
			<td>52</td>
			<td>37.14%</td>
			<td>33 ± 3</td>
		</tr>
<tr>
<td>漩涡/随机摇动</td>
			<td>49</td>
			<td>35.00%</td>
			<td>18 ± 3</td>
		</tr>
<tr>
<td>手工剥夺</td>
			<td>9</td>
			<td>6.43%</td>
			<td>36 ± 11</td>
		</tr>
<tr>
<td>热遗传学 SD</td>
			<td>15</td>
			<td>10.71%</td>
			<td>36 ± 12</td>
		</tr>
<tr>
<td>未指定</td>
			<td>15</td>
			<td>10.71%</td>
			<td>29 ± 10</td>
		</tr>
</tbody></table>表1:文献中使用的睡眠剥夺不同方法的调查。 只有116/254篇论文使用睡眠剥夺。使用每个方法的论文数量 = "论文总数"。使用每个方法的论文的分数 = "%论文 / 技术" 。评估每个方法的平均恢复长度 = "评估 Avg 恢复"。SD - 睡眠剥夺。捕捉 - 睡眠无效设备
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>SD 长度</td>
			<td>研究总额</td>
		</tr>
<tr>
<td>< 6 小时</td>
			<td>12</td>
		</tr>
<tr>
<td>6 小时</td>
			<td>23</td>
		</tr>
<tr>
<td>>6小时= < 12 小时</td>
			<td>17</td>
		</tr>
<tr>
<td>12 小时</td>
			<td>69</td>
		</tr>
<tr>
<td>>12小时= <24小时</td>
			<td>7</td>
		</tr>
<tr>
<td>24 小时</td>
			<td>19</td>
		</tr>
<tr>
<td>> 24 小时</td>
			<td>9</td>
		</tr>
<tr>
<td>慢性 SD</td>
			<td>4</td>
		</tr>
<tr>
<td>任何 SD</td>
			<td>160</td>
		</tr>
</tbody></table>表2。在不同的研究中进行的睡眠剥夺时间长短。 SD - 睡眠剥夺
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62105/62105fig01.jpg"> 图1。睡眠无效阿帕拉图斯 （捕捉） 。A） 仪器的前视图。SNAP 可在两行中容纳 8 个活动监视器;支架销将监视器固定到位。腿部可以调整，以帮助将设备定位在正确的方向。 B） 电机和弹簧的特写视图，来回摇动仪器。电机将凸轮转回"向上"位置，并压缩弹簧。从压缩释放弹簧将设备捕捉回"向下"位置。 C） "向下"位置的仪器的左侧视图。支架销约束监视器;监视器电源线插槽确保监控线保持到位。垫片有助于缓冲设备卡到"向下"位置的影响。右 - 近距离查看凸轮。 D） 设备在"向上"位置的左侧视图。右侧 - 凸轮的反顺时针旋转将设备倾斜到"向上"位置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62105/62105fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62105/62105fig02.jpg"> 图2。实验结果。A） Cs的睡眠图在实验的四天中飞: 基线日 （bs）， 睡眠剥夺日 （sd） 和两天的恢复 （rec1 和 rec2） 。B） 睡眠不足当天苍蝇的平均活动计数。苍蝇在12-24小时被剥夺了睡眠。C） 睡眠剥夺和恢复的时间过程。Cs苍蝇在 12 - 24 小时睡眠不足， 并被允许从 24 小时 - 72 小时恢复。SNAP 有效地剥夺了苍蝇的>98% 睡眠，睡眠部分恢复超过 48 小时（n = 12 苍蝇，重复测量 ANOVA 的时间，F [70，1470]+12.97，p < 10-15）。 D） 睡眠百分比恢复超过48小时。苍蝇在12小时以上恢复20%的睡眠，在实验中每天睡眠超过48小时，占睡眠巩固的36%，以白天的平均睡眠时间来衡量。与基线（p <0.05，t测试）相比，第一个恢复日睡眠更加巩固。F） 实验中每一天的睡眠巩固，以白天的最大睡眠时间来测量。与基线（p <0.05，t测试）相比，第一个恢复日睡眠更加巩固。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62105/62105fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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

睡眠剥夺是研究睡眠功能和调节的有力工具。我们描述了一个使用睡眠消除装置剥夺 嗜睡者 ，并确定剥夺引起的反弹睡眠的程度的协议。

睡眠无效装置:剥夺嗜睡者睡眠的高效方法

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

3.3K 次观看.  Washington University School of Medicine. 睡眠剥夺是研究睡眠功能和调节的有力工具。我们描述了一个使用睡眠消除装置剥夺 嗜睡者 ，并确定剥夺引起的反弹睡眠的程度的协议。

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

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.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

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

科研

神经科学

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

免疫与感染

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

行为学

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

该平衡翼：管理对酒精苍蝇

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

可调高清成像系统，用于 果蝇 成人的行为研究

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

推进睡眠障碍治疗:一种基于果 蝇的筛查方法

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

作者聚焦:使用 DAM 系统克服果蝇睡眠测量的挑战 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

睡眠无效装置:剥夺嗜睡者睡眠的高效方法

摘要

探索更多视频

此视频中的章节

含量自发活动，研究昼夜节律和睡眠参数果蝇

定量测定的免疫反应和睡眠模式

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

光偏好的检测，以研究的先天和昼夜调节感光特性

该平衡翼：管理对酒精苍蝇

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

可调高清成像系统，用于果蝇成人的行为研究

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

睡眠无效装置:剥夺嗜睡者睡眠的高效方法

摘要

探索更多视频

此视频中的章节

含量自发活动，研究昼夜节律和睡眠参数果蝇

定量测定的免疫反应和睡眠模式

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

光偏好的检测，以研究的先天和昼夜调节感光特性

该平衡翼：管理对酒精苍蝇

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

可调高清成像系统，用于 果蝇 成人的行为研究

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

使用黑腹果蝇对年龄相关睡眠障碍进行高通量小分子药物筛选

可调高清成像系统，用于果蝇成人的行为研究