Bu çalışma NIH tarafından PJS'ye 5R01NS051305-14 ve 5R01NS076980-08 hibeleri ile desteklendi.

1. Deneysel hazırlık
<ol><li>Erkek ve dişi sinekleri ayırarak şişelere dökerken sinekleri toplayın. NOT: Uyku deneyleri genellikle dişi sineklerle yapılır. Bakire dişileri toplamak önemlidir. Çift çift dişiler, verilerin analizini zorlaştıran larvalara yumurtadan çıkan yumurtaları bırakacaktır.</li>
	<li>Ev, <20'lik gruplar halinde tek bir cinsiyetin sinekleri. NOT: Konutlar sosyal olarak zenginleştirilmiş bir ortamda uçar (>50 grupları) uyku sürücüsü6,13 potansiyel olarak şaşırtıcı ribaund uykusu ölçümlerini modüle eder. Ayrıca, sosyal zenginleşmenin ardından, uyku birkaç gün içinde azalacaktır6. Bu nedenle, temel uyku, ribaund uykusunun analizini zorlaştıran kararlı değildir. Sinekleri <20'lik gruplar halinde tutmak bu potansiyel kafa karıştırıcılığı önler.</li>
	<li>Sinekleri ışık ve nem kontrollü bir ortamda 3-5 gün boyunca şişelerde tutun. NOT: Sineklerin yaşı ve olgunluğu uykuyu güçlü bir şekilde etkiler. Uyku bir günlük sineklerde yüksektir ve 3-5 günlük4yaşına kadar stabilize edilir. Sinekler tipik olarak 12 saat ışıkta tutulur: % 50 nemde 12 saat karanlık program.</li>
</ol>2. Uyku kaydı için tüplerin hazırlanması
NOT: Uyku, lokomotor aktivite monitörleri kullanılarak izlenir. Bir monitör, 5 mm çapında tüplerde ayrı ayrı barındırılanmış 32 sinek tutabilir. Tipik olarak, genotipler 16 veya 32 sinekli gruplar halinde analiz edilir.
<ol><li>Bir ucunda sinek yemi olan uygun sayıda tüp hazırlayın. NOT: Diyet ve metabolizmanın uyku33,34' ü etkilediği bilinmektedir, bu nedenle sinekleri yetiştirildikleri aynı yiyeceğe yerleştirmek özellikle önemlidir.</li>
	<li>Tüplerin ucunu balmumu ile kapatın. NOT: Uyku yoksunluğu ve ribaund beş günlük bir deneydir ve uygun şekilde kapatılmazsa yiyecekler kuruyabilir. Düzgün kapatılmış tüplerde, yiyecekler 10 gün veya daha fazla tutulabilir. Bu nedenle, tüplerin uçlarının iyi kapatıldığından emin olmak önemlidir. Bununla birlikte, sinekler ıslak yiyeceklere de yapışabilir. Böylece, deneyin başlamasından 1-2 gün önce tüplerin yapılmasına yardımcı olur.</li>
	<li>Bir aspiratör kullanarak uyku kaydı için 65 mm uzunluğunda cam tüplere tek tek uyku cihazı yerleştirin ve tüplerin ucunun bir köpük durdurucu ile takın. NOT: Sinekler, uyku kaydı için tüplere yerleştirirken asla CO2 anestezisine tekrar maruz kalmaz. Aspiratör, bir ucu cheesecloth ile kaplı kauçuk borudan yapılır ve 1 mL pipet ucuna yerleştirilir.</li>
</ol>3. Uyku kaydı
<ol><li>Uykuyu izlemek için tüplerdeki sinekleri aktivite monitörlerine yükleyin. NOT: SNAP kayaçları her ~10 sn'de bir -60° ila +60° arasında ileri geri izler. Monitörler ~5.9s için -60° seviyesinde tutulur; monitörleri tutan tepsinin -60° ila +60° ve ~1 sn'den +60° ila -60° arasında geri hareket etmesi ~2,9 sn sürer. Motora verilen voltaj ayarlanarak çevrim uzunluğu gerektiği gibi değiştirilebilir.<ol>
<li>Tüplerin aktivite monitörlerine doğru yönde yerleştirildiklerinden emin olun. Doğru yönde, sineklerin yiyeceğe itilmesini sağlamak için gıda içeren tüpün ucu SNAP'in üst kısmındadır. Ek olarak, yiyecekle ilgili son, uyku kayıt jakı ile monitörün yanındadır. Bu, etkinlik izleyicilerinin aynı anda etkinliği izlerken verimli uyku yoksunluğu için SNAP'te doğru yönlendirilmesini sağlar.</li>
	</ol>
</li>
	<li>Uykuyu izlemek için etkinlik monitörlerini kayıt odasına yerleştirin.</li>
	<li>Temel uykuyu tahmin etmek için uykuyu en az iki tam gün izleyin. NOT: Sineklerin aktivite monitörlerine yüklendiği gün, sineklerin tüplerde barındırılmaya uyum sağlamasına izin vermek için genellikle bir adaptasyon günü olarak hariç tutulur. Temel uyku, sineklerin yüklendiği günü takip eden sabahla başlayarak en az iki tam gün (48 saat) kaydedilir.</li>
	<li>Belirli bir günde ışıkların zamanından önceki gün ışıklara kadar 1 dakikalık kutulardaki sineklerin lokomotor aktivite sayılarını etkinlik kayıt yazılımı kullanarak kaydedin (örneğin, 08:00-08:00).</li>
	<li>Bir uyku35için eşik olarak 5 dakikalık hareketsizlik kullanarak özel makrolarla lokomotor etkinlik verilerinden uykuyu tahmin edin. NOT: Bir dizi uyku ölçümü, lokomotor etkinlik sayılarından hesaplanır. Bunlar arasında 24 saatin üzerinde min / s uyku, 24 saatte toplam uyku süresi, ortalama ve maksimum gündüz ve gece uykusu bout uzunlukları36.</li>
</ol>4. Uyku yoksunluğu ve iyileşme
<ol><li>Sinekler değişken süreler boyunca (örneğin, 12 saat, 24 saat ve 36 saat) uykusuz bırakılabildiğinden ve iyileşme uykusu çeşitli aralıklarla (örneğin, 6 saat, 12 saat, 24 saat ve 48 saat) değerlendirilebildiğinden, deneysel ihtıyatı ile iyileşme süresini belirler. Uyku iyileşmesi, bir uyku kazancı/ kaybı grafiği kullanılarak veya önceden belirlenmiş bir aralıkla (örneğin, 6 saat) kurtarılan yüzde uyku incelenerek görselleştirilebilir.</li>
	<li>Uyku iki temel gün boyunca sabitse, üçüncü gün, gece uykusuzluğu için etkinlik monitörlerini SNAP'e yerleştirin. NOT: Sinekler, 8 , 32,37,38uyku zamanları aralığında sağlam bir uyku ribaundu sergiler, ancak uykunun ribaund uykuyu güvenilir bir şekilde değerlendirmek için kararlı olması gerekir. Temel günler arasındaki uyku farkı 100 dakika ± olduğunda uyku sabittir.</li>
	<li>Etkinlik monitörlerinin monitör tutucu pimleri, monitör kabloları takılı ve arkası gıda ve önde plastik bariyerlerle doğru yönlendirilmiş monitörlerle sabitlendiğini unutmayın (Şekil 1). NOT: SNAP, kam her 10 sn'de bir dönecek şekilde tasarlanmıştır (Şekil 1). Plastik kesici uç, aparat "yukarı" konumundayken tüpleri geri iterek tüpleri sıfırlar. Tüplerin sıfırlanması, tüm tüplerin her döngünün başında tam hareket aralığına sahip olmasını sağlamak için önemlidir.</li>
	<li>Etkinlik monitörlerinin fişini çekin ve gece uykusuzluğundan sonra ışıklar yandıktan hemen sonra monitörleri SNAP'den çıkarın. NOT: Uyku yoksunluğunun sona erdirilmesi ve sineklerin 12 saat gece uykusuzluğundan sonra ışıklar yansın hemen iyileşmeye yerleştirilmesi kritik öneme sahiptir. Sinekleri iyileşmeye yerleştirmede 20-30 dakikalık bir gecikme bile ribaund uykusunun boyutunu engelleyebilir.</li>
	<li>İyileşme uykularını izlemek için sinekleri iki gün (48 saat) rahatsız edilmeyecekleri bir kayıt odasına yerleştirin. NOT: Kayıt odası diğer deneyler için kullanılıyorsa, geri kazanıcı sinekleri uyarmamak için ekstra özen uygulanmalıdır.</li>
	<li>Kaybedilen uyku miktarını hesaplayın. Her bir sinek için, uyku yoksunluğu sırasında elde edilen uyku ile taban çizgisi sırasında ilgili saat arasındaki saatlik farkı hesaplayın; kaybedilen toplam uykuyu hesaplamak için saatlik farkları toplayın.</li>
	<li>Kurtarılan uyku miktarını hesaplayın. Her bir sinek için, kurtarma sırasında elde edilen uyku ile taban çizgisi sırasında ilgili saat arasındaki saatlik farkı hesaplayın; kazanılan toplam uykuyu hesaplamak için saatlik farkları toplayın. NOT: Bir sineğin gerçekten uykusuz olup olmadığı ampiriktir. Bu nedenle, deneyci kaybedilen uyku yüzdesini incelemelidir. Sinek yeterli miktarda uyku kaybetmediyse, analizden hariç tutulabilir. Bu diğer uyku yoksunluğu yaklaşımları için gerekli olsa da, SNAP için nadiren gereklidir. Daha yaygın olarak, uyku yoksunluğu başlatıl işlemden önce belirli bir sinekte uyku sabit olmayabilir. Uyku stabil değilse, homeostaz hesaplanamaz. Uyku yoksunluğunun başlatılmasından önce hesaplanan maksimum ± 100 dakikalık uyku farkını dahil etme adayı olarak kabul ediyoruz. Bazen, bireysel bir sinek uykusu 24 saat boyunca eşit olmayan bir şekilde dağıtılır (örneğin, bazı bireyler gün boyunca uyku kotalarının% 60-70'ini alabilir ve böylece geceleri 12 saat boyunca yoksun bırakıldığında 24 saat uyku kotalarının sadece küçük bir oranını kaybedebilir). Bu sinekler ayrı ayrı değerlendirilebilir.</li>
	<li>Her genotip için kurtarma süresinin 12 h, 24 h ve 48 h üzerinde kurtarılan ortalama uyku yüzdesini (taban çizgisine göre) hesaplayın.</li>
	<li>Uyku verilerinden, taban çizgisi ve her genotip için kurtarma gün ortalama ve maksimum gündüz uyku bout uzunluğunu hesapla. NOT: Sineklerde ribaund uykusu, iyileşme günlerinde artan uyku miktarı ve artan uyku derinliği ile karakterizedir. Uyku konsolidasyonu, uyku derinliğinin ölçüsü olarak kullanılır. Uyarılma eşikleri uyku derinliğinin bir ölçüsü olarak da kullanılabilir.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>

Yazarların açıklayacak bir şeyi yok.

Drosophila'da uyku bağımsız olarak 2000 yılında, iki grup tarafından karakterize edildi4,5. Bu öncü çalışmalarda sinekler nazik bir şekilde ele alarak uykudan mahrum bırakıldı (yani el yoksunluğu) ve gece uykusuzluğuna karşı sağlam bir homeostatik tepki sergiledikleri gösterilmiştir. Daha da önemlisi, herhangi bir uyku yoksunluğu deneyi ile, hayvanı uyanık tutmak için kullanılan yöntemin potansiyel şaşırtıcı etkilerini k...

Uyku hayvanlarda evrensele yakındır, ancak işlevi belirsizliğini korumaktadır. Uyku homeostazisi, uyku yoksunluğundan sonra uykudaki telafi edici artış, bir dizi hayvanda uyku durumlarını karakterize etmek için kullanılan uykunun belirleyici bir özelliğidir1, 2,3,4,5.
Sinekte uyku, uyku kaybına sağlam bir homeostatik yanıt da dahil olmak üzere insan uykusu ile birçok benzerliğe sahiptir4,5. Sinekte çok sayıda uyku çalışması, hem uzun uyanmadan tahakkuk eden olumsuz sonuçları inceleyerek uyku fonksiyonunu çıkarmak hem de uykunun homeostatik düzenlemesini kontrol eden nörobiyolojik mekanizmaları belirleyerek uyku düzenlemesini anlamak için uyku yoksunluğunu kullanmıştır. Böylece uykusuz sineklerin öğrenme ve hafızada bozukluklar gösterdiği gösterilmiştir6,7,8,9,10,11,12, yapısal plastisite13,14,15, görsel dikkat16, nöronal yaralanmadan iyileşme17,18, çiftleşme ve agresif davranışlar19, 20, hücre çoğalması21ve oksidatif strese yanıtlar22,23 birkaç isim. Ayrıca, rebound uykuyu kontrol eden nörobiyolojik mekanizmalar üzerine yapılan araştırmalar, uyku homeostat 8 , 9 , 23 , 24,25,26,27,28,29'ı oluşturan nöronal makineler hakkında kritik içgörüler sağladı. . Son olarak, sağlıklı hayvanlarda uyku işlevine dair temel içgörüleri ortaya çıkarmanın yanı sıra, uyku yoksunluğu çalışmaları da hastalıklı durumlarda uyku fonksiyonu hakkında içgörüleri bilgilendirmiş30,31.
Uyku yoksunluğu inkar edilemez derecede güçlü bir araç olsa da, herhangi bir uyku yoksunluğu deneyi ile, genişletilmiş uyanmadan kaynaklanan fenotipleri, hayvanı uyanık tutmak için kullanılan uyaranın neden olduğulardan ayırt etmek önemlidir. El yoksunluğu veya nazik kullanım ile uyku yoksunluğu, genellikle minimal bozucu uyku yoksunluğu için standardı belirlemek olarak kabul edilir. Burada, Uyku Nulllaştırma Aparatı'nı (SNAP) kullanarak sinekleri uykudan yoksun bırakmak için bir protokol açıklıyoruz. SNAP, her 10'larda bir sineklere mekanik bir uyaran sağlayan, negatif jeotaksiyi indükle ederek sinekleri uyanık tutan bir cihazdır (Şekil 1). SNAP, yüksek uyku sürücüsüne sahip sineklerde bile gece uykusunun% >98'inden verimli bir şekilde mahrum eder8,32. SNAP patlamaya duyarlı sineklerde kalibre edilmiştir, SNAP'teki sineklerin ajitasyonu sineklere zarar vermez; SNAP ile uyku yoksunluğu, el yoksunluğu ile elde edilenle karşılaştırılabilir bir ribaund sağlar7. Snap böylece uyandırıcı uyaranın etkilerini kontrol ederken sinekleri mahrum bırakmak için sağlam bir yöntemdir.

<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

Uyku homeostazisi, uyku kaybının ardından gözlenen uykudaki artış, hayvanlar alemi boyunca uykuyu tanımlamak için kullanılan belirleyici kriterlerden biridir. Sonuç olarak, uyku yoksunluğu ve uyku kısıtlaması, uyku işlevi hakkında fikir vermek için yaygın olarak kullanılan güçlü araçlardır. Bununla birlikte, uyku yoksunluğu deneyleri, yoksunluk uyaranının fizyoloji ve davranışta gözlenen değişikliklerin nedeni olabileceği için doğası gereği sorunludur. Buna göre, başarılı uyku yoksunluğu teknikleri hayvanları uyanık tutmalı ve ideal olarak, çok sayıda istenmeyen sonuç doğurmadan sağlam bir uyku ribaundu ile sonuçlanmalıdır. Burada, Drosophila melanogasteriçin bir uyku yoksunluğu tekniğini tarif ediyoruz. Uyku Nullifiye Aparatı (SNAP), negatif jeotaksisi teşvik etmek için her 10'da bir uyaran uygular. Uyaran öngörülebilir olsa da, SNAP, yüksek uyku sürücüsüne sahip sineklerde bile gece uykusunun% 95'> etkili bir şekilde önler. Daha da önemlisi, sonraki homeostatik yanıt, el yoksunluğu kullanılarak elde edilene çok benzer. Uyaranların zamanlaması ve aralığı, uyku kaybını en aza indirmek ve böylece uyaranın fizyoloji ve davranış üzerindeki spesifik olmayan etkilerini incelemek için değiştirilebilir. SNAP, uyku kısıtlaması ve uyarılma eşiklerini değerlendirmek için de kullanılabilir. SNAP, uyku işlevini daha iyi anlamak için kullanılabilecek güçlü bir uyku bozulması tekniğidir.

Kanton S (Cs) vahşi tip suş olarak kullanılmıştır. Sinekler 12 saat ışıkta tutuldu: 12 saat karanlık program ve gece boyunca 12 saat uykusuz kaldı. Temel günde Cs sineklerinin uyku profillerinin incelenmesi (bs), uyku yoksunluğu günü (sd) ve iki iyileşme günü (rec1 ve rec2) (Şekil 2A),sineklerin SNAP'te etkili bir şekilde uykusuz kaldığını ve literatürde gözlemlenen raporlarla tutarlı olarak gün içinde iyileştiğini göstermekted...

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

Uyku yoksunluğu, uyku fonksiyonunu ve düzenlemesini araştırmak için güçlü bir araçtır. Uyku Nullifiye Aparatı'nı kullanarak Drosophila'yı uykudan yoksun bırakmak ve yoksunluğun neden olduğu ribaund uykusunun kapsamını belirlemek için bir protokol açıklıyoruz.

Uyku Nullifiye Aparatı: Drosophila'dan Yoksun Uykunun Son Derece Verimli Bir Yöntemi

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

3.3K Görüntüleme.  Washington University School of Medicine. Uyku yoksunluğu, uyku fonksiyonunu ve düzenlemesini araştırmak için güçlü bir araçtır. Uyku Nullifiye Aparatı'nı kullanarak Drosophila'yı uykudan yoksun bırakmak ve yoksunluğun neden olduğu ribaund uykusunun kapsamını belirlemek için bir protokol açıklıyoruz.

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

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

İlaç Takviye için yeni cihaz ve Yöntem

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

Nörobilim

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

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

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

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Sirkadiyen Rhythms Eğitim ve Parametreler Uyku Lokomotor Aktivitesi Assaying Drosophila</em

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

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Yüksek zenginleştirilmiş Nöronal Kültürlerin Üretimi için Kortikal Doku Blokları, Kriyoprezervasyon

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

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.

Toplam RNA İzolasyonu ve Alt Nesil Sıralama ve Kaynak gösterilmeden kullanılamaz Ayrık Fare Beyin Bölgeler Mikrodisseksiyon Sigara Lazer Yakalama Mikroskopi Yaklaşım

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.

Eşzamanlı Elektroensefalografi, Kemirgen Serebral Korteks Laktat Konsantrasyonu ve Nöronal Faaliyet Optogenetic Manipülasyon Gerçek zamanlı ölçüm

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.

Sprague-Dawley Sıçan nodoz Ganglionlar Enjeksiyon Bir Yöntem

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

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Kapalı Başkanı Travmatik Beyin yaralayabilecek Bir Yöntem<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

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

Farelerde Uyku Ölçüm Basım Kayıt Prosedürü

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.

Farelerde uyku/uyanıklık durumları Izleme sırasında nöral devrelerin optogenetik manipülasyon

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.

Uyku Nullifiye Aparatı: Drosophila'dan Yoksun Uykunun Son Derece Verimli Bir Yöntemi

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