This work was supported by NIH grants 5R01NS051305-14 and 5R01NS076980-08 to PJS.

1. Experimental preparation
<ol>
	<li>Collect flies as they eclose into vials, separating male and female flies. 
	NOTE: Sleep experiments are commonly conducted with female flies. It is important to collect virgin females. Mated females will lay eggs that hatch into larvae complicating the analysis of the data.</li>
	<li>House flies of a single sex in groups of &#60;20. 
	NOTE: Housing flies in a socially enriched environment (groups of &#62;50) modulates sleep drive6,<sup ...

<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>

Sleep in Drosophila was independently characterized in 2000, by two groups4,5. In these pioneering studies, flies were deprived of sleep by gentle handling (i.e., hand deprivation) and shown to exhibit a robust homeostatic response to overnight sleep deprivation. Importantly, with any sleep deprivation experiment it is crucial to control for potential confounding effects of the method used to keep the animal awake. Hand deprivation studies set the benchm...

Sleep is near universal in animals, yet its function remains unclear. Sleep homeostasis, the compensatory increase in sleep following sleep deprivation, is a defining property of sleep, that has been used to characterize sleep states in a number of animals1,2,3,4,5.
Sleep in the fly has many similarities with human sleep, including a robust homeostatic response to sleep loss4,5. Numerous studies of sleep in the fly have used sleep deprivation both to infer sleep function by examining the adverse consequences that accrue from extended waking, and to understand sleep regulation by determining the neurobiological mechanisms controlling the homeostatic regulation of sleep. Thus sleep deprived flies were shown to exhibit impairments in learning and memory6,7,8,9,10,11,12, structural plasticity13,14,15, visual attention16, recovery from neuronal injury17,18, mating and aggressive behaviors19,20, cell proliferation21, and responses to oxidative stress22,23 to name a few. Further, investigations into the neurobiological mechanisms controlling rebound sleep have yielded critical insights into the neuronal machinery that constitutes the sleep homeostat8,9,23,24,25,26,27,28,29. Finally, in addition to revealing fundamental insights into sleep function in healthy animals, sleep deprivation studies have also informed insights into sleep function in diseased states30,31.
While sleep deprivation is undeniably a powerful tool, with any sleep deprivation experiment, it is important to distinguish phenotypes that result from extended waking, from those induced by the stimulus used to keep the animal awake. Sleep deprivation by hand deprivation or gentle handling, is generally regarded as setting the standard for minimally disruptive sleep deprivation. Here we describe a protocol for sleep depriving flies using the Sleep Nullifying Apparatus (SNAP). The SNAP is a device that delivers a mechanical stimulus to flies every 10s, keeping flies awake by inducing negative geotaxis (Figure 1). The SNAP efficiently deprives flies of &#62;98% of night-time sleep, even in flies with high sleep drive8,32. The SNAP has been calibrated on bang sensitive flies, agitation of flies in the SNAP does not harm flies; sleep deprivation with the SNAP induces a rebound comparable with that obtained by hand deprivation7. The SNAP is thus a robust method to sleep deprive flies while controlling for the effects of the arousing stimulus.

<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

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents &#62;95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

Canton S (Cs) was used as a wild-type strain. Flies were maintained on a 12 h light: 12 h dark schedule, and sleep deprived for 12 hours overnight. Inspection of the sleep profiles of Cs flies on the baseline day (bs), sleep deprivation day (sd), and two recovery days (rec1 and rec2) (Figure 2A) suggests that flies were effectively sleep deprived in the SNAP, and recovered sleep during the day consistent with observed reports in the literature<sup class="xref...

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

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation.

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

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

Sleep homeostasis, the increase in sleep following sleep loss, is a defining characteristic of sleep. Sleep deprivation and sleep restriction are thus powerful tools to study sleep regulation and function. This protocol efficiently sleep deprives and sleep restricts flies while minimizing potential confounds.SNAP deprives flies of greater than 95%of sleep even in flies with high sleep drive. Importantly, agitation of flies with the SNAP does not harm flies and induce a rebound comparable to that obtained with hand deprivation, which is a standard for minimally disruptive sleep deprivation. Visualizing how the SNAP keeps flies awake will help investigators use and optimize this protocol.Begin by collecting eclosing flies into vials, separating males and females. House the flies in groups of less than 20 and keep them for three to five days in a light and humidity-controlled environment. Prepare an appropriate number of tubes with fly food at one end and seal the end with wax.Individually place awake behaving flies into the tubes using an aspirator and plug the tubes with a foam stopper. Load the tubes into activity monitors to monitor sleep, making sure that the tubes are placed in the correct orientation. The end of the tube of food should be at the top of the SNAP to ensure that flies do not get pushed into the food.Place activity monitors in the recording chamber and monitor sleep for at least two full days to estimate baseline sleep. Save locomotor activity counts of flies in one-minute bins from the time of lights on a given day to lights on the previous day using activity recording software. Estimate sleep from the locomotor activity data with custom macros using five minutes of inactivity as the threshold for a bout of sleep.If sleep is stable over the two baseline days, place activity monitors into the SNAP for overnight sleep deprivation on the third day. Make sure activity monitors are secured in place with monitor holder pins. The monitor cords are plugged in and the monitors oriented correctly.Once the lights go on after overnight sleep deprivation, unplug activity monitors and take them out of the SNAP immediately. Place the flies in a recording chamber where they will be undisturbed for two days to monitor recovery sleep. For each individual fly, calculate the hourly difference between sleep obtained during sleep deprivation and the corresponding hour during baseline, then sum the hourly differences to calculate total sleep lost.Next, calculate the hourly difference between sleep obtained during recovery and the corresponding hour during baseline, then sum the hourly differences to calculate total sleep gained. Calculate the average percentage of sleep recovered over 12, 24, and 48 hours of the recovery period for each genotype. Finally, compute the average and maximum daytime sleep bout length on baseline and recovery days for each genotype.Flies were sleep deprived in the SNAP and recovered sleep during the day. The effectiveness of SNAP in keeping flies awake was demonstrated with the high activity exhibited by flies during sleep deprivation. To quantitatively estimate the effectiveness of sleep deprivation and of recovery, sleep lost during deprivation and then regained in the recovery days was calculated for each individual fly.Importantly, there was no significant change in baseline sleep between the deprivation day and the baseline day, indicating that sleep is stable in these flies. The SNAP effectively deprived flies of over 98%of their nighttime sleep. Flies recovered approximately 20%of their sleep in the first 12 hours and did not recover additional sleep during the night.They began to recover sleep the following day and recovered 36%of their sleep over 48 hours. Sleep homeostasis is characterized by both increased sleep duration and by increased sleep depth during the recovery period. Daytime sleep consolidation is commonly used as a readout of sleep depth.Sleep consolidation can be assessed as the average sleep bout duration over the entire day. However, as sleep pressure is dissipated during recovery, the average sleep bout duration is reduced. Changes in the maximum sleep bout duration can provide a more sensitive metric.This procedure can be easily modified to minimize sleep loss, thus control for non-specific effects of the stimulus. SNAP can be configured to restrict sleep, thus mimicking chronic sleep loss in humans. It can also be used to measure arousal thresholds.Sleep deprivation using the SNAP has yielded important insights into sleep function through studies that examine the negative consequences of sleep loss. By identifying manipulations that interfere with the expression of rebound sleep, sleep deprivation with the SNAP has also helped to elucidate homeostatic mechanisms that regulate sleep.

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3.2K ビュー.  Washington University School of Medicine. Sleep homeostasis, the increase in sleep following sleep loss, is a defining characteristic of sleep. Sleep deprivation and sleep restriction are thus powerful tools to study sleep regulation and function. This protocol efficiently sleep deprives and sleep restricts flies while minimizing potential confounds.SNAP deprives flies of greater than 95%of sleep even in flies with high sleep drive. Importantly, agitation of flies with the SNAP does not harm flies and induce a rebound comparab...

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

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by...

quantitative measurement of the immune response and sleep in drosophila

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,...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

high-throughput small molecule drug screening for age-related sleep disorders using drosophila melanogaster

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

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

Advancing Sleep Disorder Treatments: A &lt;em&gt;Drosophila&lt;/em&gt;-Based Screening Approach

drug screening method for sleep regulation in an elderly drosophila melanogaster model

Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model  

Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model

establishing a device for sleep deprivation in mice

Establishing a Device for Sleep Deprivation in Mice

The present protocol outlines a method for setting up a cost-effective rocker platform-based device used for inducing sleep deprivation in mice. This device has proven to be effective in causing disruptions in electroencephalogram (EEG)-evidenced sleep patterns, as well as inducing metabolic and molecular changes associated with sleep...

assaying locomotor activity to study circadian rhythms and sleep parameters in drosophila

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...

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

analyzing the effect of reserpine on the sleep behavior of drosophila melanogaster using scamp2020 data package in matlab

Analyzing the Effect of Reserpine on the Sleep Behavior of Drosophila melanogaster Using SCAMP2020 Data Package in MATLAB  

Analyzing the Effect of Reserpine on the Sleep Behavior of Drosophila melanogaster Using SCAMP2020 Data Package in MATLAB

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

noninvasive, high-throughput determination of sleep duration in rodents

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human...

eye tracking, cortisol, and a sleep vs. wake consolidation delay: combining methods to uncover an interactive effect of sleep and cortisol on memory

Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory

We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing &#8211; methods that can be used with both healthy and clinical...

automated measurements of sleep and locomotor activity in mexican cavefish

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish...

acupoint application combined with ear plaster therapy for treating sleep disorders with acute exacerbation of chronic obstructive pulmonary disease

Author Spotlight: Traditional Chinese Medicine for Sleep Disorders in Acute COPD &#8212; A Safe, Cost-Effective Approach

The present protocol describes the specific process and efficacy of the acupoint application combined with ear plaster therapy for sleep disorders in a patient with acute exacerbation of chronic obstructive pulmonary disease, including medical devices, selection of acupoints, operating methods, and posttreatment...

Acupoint and Ear Plaster Therapy for Treating Sleep Disorders in AECOPD Patients

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chronic sleep deprivation in mouse pups by means of gentle handling

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and...

drug-induced sleep endoscopy (dise) with target controlled infusion (tci) and bispectral analysis in obstructive sleep apnea

Drug-Induced Sleep Endoscopy DISE with Target Controlled Infusion TCI and Bispectral Analysis in Obstructive Sleep Apnea

The aim of this study was to establish a standardized protocol for sleep endoscopy to differentiate obstruction patterns in obstructive sleep apnea (OSA). Target-controlled infusion (TCI) of the sedative was combined with real-time monitoring of the depth of sedation using bispectral...

Drug-Induced Sleep Endoscopy (DISE) with Target Controlled Infusion (TCI) and Bispectral Analysis in Obstructive Sleep Apnea

multi-modal home sleep monitoring in older adults

Multi-Modal Home Sleep Monitoring in Older Adults

Here, we present a protocol to improve the quality of data from home sleep testing by providing a method to enhance instructions through a structured participant visit. This protocol includes the implementation of a step-by-step educational manual with photos to ensure proper placement of...

lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized...

intellisleepscorer, a software package with a graphic user interface for mice automated sleep stage scoring

Author Spotlight: IntelliSleepScorer &#8212; A High-Accuracy, Accessible GUI Software for Automated Sleep Stage Scoring in Mice and its Application in Psychiatric Research

We present a software package with a graphic user interface for researchers without coding experience to score sleep stages in mice with a simple download and operation.

IntelliSleepScorer, a Software Package with a Graphic User Interface for Mice Automated Sleep Stage Scoring

polygraphic recording procedure for measuring sleep in mice

Polygraphic Recording Procedure for Measuring Sleep in Mice

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

collecting sleep, circadian, fatigue, and performance data in complex operational environments

Collecting Sleep, Circadian, Fatigue, and Performance Data in Complex Operational Environments

Sleep loss and circadian misalignment contribute to numerous operational accidents and incidents. The effectiveness of countermeasures and work scheduling designs aimed at mitigating fatigue can be challenging to evaluate in operational environments. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in complex operational...

measuring neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood

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

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...

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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