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

Este trabalho foi apoiado pelas bolsas NIH 5R01NS051305-14 e 5R01NS076980-08 para PJS.

1. Preparação experimental
<ol><li>Recolher moscas enquanto se fecham em frascos, separando moscas machos e fêmeas. NOTA: Experimentos de sono são comumente realizados com moscas fêmeas. É importante coletar fêmeas virgens. Fêmeas acasaladas colocarão ovos que eclodem em larvas complicando a análise dos dados.</li>
	<li>House voa de um único sexo em grupos de <20. NOTA: A habitação voa em um ambiente socialmente enriquecido (grupos de >50) modula a unidade de sono6,...

<ol>
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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. 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>

O sono em Drosophila foi caracterizado independentemente em 2000, por dois grupos4,5. Nestes estudos pioneiros, as moscas foram privadas do sono por manuseio suave (ou seja, privação de mão) e mostraram uma resposta homeostática robusta à privação do sono durante a noite. É importante ressaltar que, com qualquer experimento de privação do sono, é crucial controlar os potenciais efeitos de confusão do método usado para manter o animal acordad...

O sono é quase universal em animais, mas sua função permanece incerta. A homeostase do sono, o aumento compensatório do sono após a privação do sono, é uma propriedade definidora do sono, que tem sido usada para caracterizar estados de sono em um número de animais1,2,3,4,5.
O sono na mosca tem muitas semelhanças com o sono humano, incluindo uma resposta homeostática robusta à perda de sono4,5. Inúmeros estudos de sono na mosca têm usado a privação do sono tanto para inferir a função do sono, examinando as consequências adversas que se acumulam a partir da vigília prolongada, quanto para entender a regulação do sono, determinando os mecanismos neurobiológicos que controlam a regulação homeostática do sono. Assim, as moscas privadas de sono apresentaram prejuízos no aprendizado e na memória6,7,8,9,10,11,12, plasticidade estrutural13,14,15, atenção visual16, recuperação da lesão neuronal17,18, acasalamento e comportamentos agressivos19, 20, proliferação celular21, e respostas ao estresse oxidativo22,23 para citar alguns. Além disso, as investigações sobre os mecanismos neurobiológicos que controlam o sono de recuperação produziram insights críticos sobre a maquinaria neuronal que constitui o homeostat do sono8,9,23,24,25,26,27,28,29 . Finalmente, além de revelar insights fundamentais sobre a função do sono em animais saudáveis, estudos de privação do sono também informaram insights sobre a função do sono em estados doentes30,31.
Embora a privação do sono seja inegavelmente uma ferramenta poderosa, com qualquer experimento de privação do sono, é importante distinguir fenótipos que resultam de um aumento prolongado, daqueles induzidos pelo estímulo usado para manter o animal acordado. A privação do sono por privação manual ou manuseio suave, é geralmente considerada como estabelecendo o padrão para privação de sono minimamente disruptiva. Aqui descrevemos um protocolo para o sono privando moscas usando o Aparelho de Anulação do Sono (SNAP). O SNAP é um dispositivo que fornece um estímulo mecânico para moscas a cada 10 anos, mantendo as moscas acordadas induzindo geotaxis negativos(Figura 1). O SNAP priva eficientemente moscas de >98% do sono noturno, mesmo em moscas com alta movimentação de sono8,32. O SNAP foi calibrado em moscas sensíveis ao estrondo, a agitação das moscas no SNAP não prejudica as moscas; a privação do sono com o SNAP induz um rebote comparável ao obtido por privação manual7. O SNAP é, portanto, um método robusto para dormir privar moscas enquanto controla os efeitos do estímulo estimulante.

<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

A homeostase do sono, o aumento do sono observado após a perda do sono, é um dos critérios definidores utilizados para identificar o sono em todo o reino animal. Como consequência, a privação do sono e a restrição do sono são ferramentas poderosas que são comumente usadas para fornecer insights sobre a função do sono. No entanto, os experimentos de privação do sono são inerentemente problemáticos, na prática, o estímulo à privação em si pode ser a causa das mudanças observadas na fisiologia e no comportamento. Assim, técnicas bem sucedidas de privação do sono devem manter os animais acordados e, idealmente, resultar em uma recuperação robusta do sono sem também induzir um grande número de consequências não intencionais. Aqui, descrevemos uma técnica de privação do sono para Drosophila melanogaster. O Aparelho de Anulação do Sono (SNAP) administra um estímulo a cada 10 anos para induzir geotaxis negativos. Embora o estímulo seja previsível, o SNAP efetivamente evita >95% do sono noturno mesmo em moscas com alta movimentação de sono. É importante ressaltar que a resposta homeostática subsequente é muito semelhante à alcançada usando privação manual. O tempo e o espaçamento dos estímulos podem ser modificados para minimizar a perda de sono e, assim, examinar efeitos não específicos do estímulo sobre fisiologia e comportamento. O SNAP também pode ser usado para restrição de sono e para avaliar limiares de excitação. O SNAP é uma poderosa técnica de interrupção do sono que pode ser usada para entender melhor a função do sono.

Canton S (Cs) foi usado como uma cepa tipo selvagem. As moscas foram mantidas em uma luz de 12 h: horário escuro de 12h e privação de sono por 12 horas durante a noite. A inspeção dos perfis de sono das moscas de Cs no dia da linha de base (bs), dia de privação do sono (sd) e dois dias de recuperação (rec1 e rec2)(Figura 2A) sugere que as moscas foram efetivamente privadas de sono no SNAP, e recuperaram o sono durante o dia consistente com os relatos...

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

A privação do sono é uma ferramenta poderosa para investigar a função e a regulação do sono. Descrevemos um protocolo para dormir privar Drosophila usando o Aparelho de Anulação do Sono, e determinar a extensão do sono de recuperação induzido pela privação.

O aparelho de anulação do sono: um método altamente eficiente de privação do sono 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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The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

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