Name: the sleep nullifying apparatus: a highly efficient method of sleep depriving drosophila
Uploaded: 2020-12-14T15:00:00
Description: Watch this Scientific Journal Video about The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila at JoVE.com

Dit werk werd ondersteund door NIH-subsidies 5R01NS051305-14 en 5R01NS076980-08 aan PJS.

1. Experimentele voorbereiding
<ol><li>Verzamel vliegen terwijl ze in flesjes eclose komen en mannelijke en vrouwelijke vliegen scheiden. OPMERKING: Slaapexperimenten worden vaak uitgevoerd met vrouwelijke vliegen. Het is belangrijk om maagdelijke vrouwtjes te verzamelen. Gepaarde vrouwtjes leggen eieren die uitkomen in larven die de analyse van de gegevens bemoeilijken.</li>
	<li>Huisvliegen van een enkel geslacht in groepen van <20. OPMERKING: Huisvestingsvliegen in een sociaal verrijkte omgeving (groepen van >...

<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. 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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. 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Slaap in Drosophila werd onafhankelijk gekenmerkt in 2000, door twee groepen4,5. In deze baanbrekende studies werden vliegen beroofd van slaap door zachte behandeling (d.w.z. handgebrek) en bleek ze een robuuste homeostatische reactie op nachtelijk slaapgebrek te vertonen. Belangrijk is dat het bij elk slaapgebreksexperiment cruciaal is om te controleren op mogelijke verstorende effecten van de methode die wordt gebruikt om het dier wakker te houden. Han...

Slaap is bijna universeel bij dieren, maar de functie ervan blijft onduidelijk. Slaaphomeostase, de compenserende toename van slaap na slaapgebrek, is een bepalende eigenschap van slaap, die is gebruikt om slaaptoestanden te karakteriseren bij een aantal dieren1,2,3,4,5.
Slaap in de vlieg heeft veel overeenkomsten met de menselijke slaap, waaronder een robuuste homeostatische reactie op slaapverlies4,5. Talrijke studies van slaap in de vlieg hebben slaapgebrek gebruikt, zowel om de slaapfunctie af te leiden door de nadelige gevolgen van langdurig ontwaken te onderzoeken, als om slaapregulatie te begrijpen door de neurobiologische mechanismen te bepalen die de homeostatische regulatie van slaap regelen. Zo werd aangetoond dat slaaparme vliegen beperkingen vertonen in leren en geheugen6,7,8,9,10,11,12,structurele plasticiteit13,14,15,visuele aandacht16,herstel van neuronaal letsel17,18,paring en agressief gedrag19, 20,celproliferatie 21, en reacties op oxidatieve stress22,23 om er maar een paar te noemen. Verder hebben onderzoeken naar de neurobiologische mechanismen die de reboundslaap regelen kritische inzichten opgeleverd in de neuronale machinerie die de slaaphomeostat8,9,23,24,25,26,27,28,29 vormt . Ten slotte hebben slaapgebreksstudies, naast het onthullen van fundamentele inzichten in de slaapfunctie bij gezonde dieren, ook inzichten verkregen in de slaapfunctie in zieke toestanden30,31.
Hoewel slaapgebrek ontegenzeggelijk een krachtig hulpmiddel is, is het bij elk slaapgebreksexperiment belangrijk om fenotypen te onderscheiden die het gevolg zijn van langdurig wakker worden, van fenotypen die worden veroorzaakt door de stimulus die wordt gebruikt om het dier wakker te houden. Slaaptekort door handgebrek of zachte behandeling, wordt over het algemeen beschouwd als het bepalen van de norm voor minimaal verstorend slaapgebrek. Hier beschrijven we een protocol voor slaaprovende vliegen met behulp van het Sleep Nullifying Apparatus (SNAP). De SNAP is een apparaat dat elke 10s een mechanische stimulus aan vliegen levert, waardoor vliegen wakker blijven door negatieve geotaxis te induceren(figuur 1). De SNAP berooft vliegen efficiënt van >98% van de nachtrust, zelfs bij vliegen met een hoge slaapaandrijving8,32. De SNAP is gekalibreerd op banggevoelige vliegen, agitatie van vliegen in de SNAP is niet schadelijk voor vliegen; slaapgebrek met de SNAP induceert een rebound vergelijkbaar met die verkregen door handdeprivatie7. De SNAP is dus een robuuste methode om vliegen te slapen en tegelijkertijd de effecten van de prikkelende stimulus te beheersen.

<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

Slaaphomeostase, de toename van slaap waargenomen na slaapverlies, is een van de bepalende criteria die worden gebruikt om slaap in het hele dierenrijk te identificeren. Als gevolg hiervan zijn slaapgebrek en slaapbeperking krachtige hulpmiddelen die vaak worden gebruikt om inzicht te geven in de slaapfunctie. Niettemin zijn slaapgebreksexperimenten inherent problematisch omdat de deprivatieprikkel zelf de oorzaak kan zijn van waargenomen veranderingen in fysiologie en gedrag. Dienovereenkomstig moeten succesvolle slaapgebrekstechnieken dieren wakker houden en idealiter resulteren in een robuuste slaaprebound zonder ook een groot aantal onbedoelde gevolgen te veroorzaken. Hier beschrijven we een slaaptekorttechniek voor Drosophila melanogaster. Het Sleep Nullifying Apparatus (SNAP) dient elke 10s een stimulus toe om negatieve geotaxis te induceren. Hoewel de stimulus voorspelbaar is, voorkomt de SNAP effectief >95% van de nachtrust, zelfs bij vliegen met een hoge slaapdrift. Belangrijk is dat de daaropvolgende homeostatische respons sterk lijkt op die bereikt met behulp van handdeprivatie. De timing en afstand van de stimuli kan worden aangepast om slaapverlies te minimaliseren en zo niet-specifieke effecten van de stimulus op fysiologie en gedrag te onderzoeken. De SNAP kan ook worden gebruikt voor slaapbeperking en om opwindingsdrempels te beoordelen. De SNAP is een krachtige slaapverstoringstechniek die kan worden gebruikt om de slaapfunctie beter te begrijpen.

Canton S (Cs) werd gebruikt als een wild-type stam. Vliegen werden gehouden op een 12 uur licht: 12 uur donker schema, en slaap beroofd voor 12 uur 's nachts. Inspectie van de slaapprofielen van Cs-vliegen op de baselinedag (bs), slaapgebreksdag (sd) en twee hersteldagen (rec1 en rec2) (Figuur 2A) suggereert dat vliegen effectief slaapgebrek hadden in de SNAP en overdag herstelden in overeenstemming met waargenomen rapporten in de literatuur...

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

Slaaptekort is een krachtig hulpmiddel om de slaapfunctie en -regulatie te onderzoeken. We beschrijven een protocol om Drosophila te ontnemen met behulp van het Slaapvernietigend Apparaat en om de mate van rebound slaap veroorzaakt door deprivatie te bepalen.

Het slaapvernietigende apparaat: een zeer efficiënte methode om drosophila te beroven

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.

Research

JoVE Journal

Neuroscience

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

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