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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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>

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?...

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 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...

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

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

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.

新型仪器和药物加固方法

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

科研

神经科学

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.

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) ...

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.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

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.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

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.

结状神经节注射液只SD大鼠的方法

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.

印刷业记录程序在小鼠体内测量睡眠

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.

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

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.

如何获得新生儿中可靠的视觉事件相关潜力

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.

测量幼儿期午睡期间睡眠依赖记忆巩固背后的神经机制

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.

使用腺相关病毒在小鼠中瞄准科菲林操纵的大脑皮层区域的电皮质记录

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

Methods Article

睡眠无效装置：剥夺嗜睡者睡眠的高效方法