Questo lavoro è stato supportato dalle sovvenzioni NIH 5R01NS051305-14 e 5R01NS076980-08 a PJS.

1. Preparazione sperimentale
<ol><li>Raccogli le mosche mentre si chiude in fiale, separando le mosche maschili e femminili. NOTA: gli esperimenti sul sonno sono comunemente condotti con mosche femmine. È importante raccogliere femmine vergini. Le femmine accoppiate deporteranno uova che si schiudono in larve complicando l'analisi dei dati.</li>
	<li>Mosche domestiche di un solo sesso in gruppi di <20. NOTA: l'alloggiamento vola in un ambiente socialmente arricchito (gruppi di >50) modula il sonno6,13 misurazioni potenzialmente confondenti del sonno di rimbalzo. Inoltre, a seguito dell'arricchimento sociale, il sonno diminuirà in pochi giorni6. Pertanto, il sonno basale non è stabile complicando l'analisi del sonno di rimbalzo. Mantenere le mosche in gruppi di <20 evita questo potenziale confusione.</li>
	<li>Tenere le mosche in fiale per 3-5 giorni in un ambiente controllato dalla luce e dall'umidità. NOTA: L'età e la maturità delle mosche influenzano fortemente il sonno. Il sonno è alto nelle mosche di un giorno e si stabilizza entro 3-5 giorni di età4. Le mosche sono in genere mantenute su una luce di 12 ore: 12 ore di buio al 50% di umidità.</li>
</ol>2. Preparazione dei tubi per la registrazione del sonno
NOTA: il sonno viene monitorato utilizzando monitor di attività locomotoria. Un monitor può contenere 32 mosche alloggiate singolarmente in tubi di diametro 5 mm. Tipicamente, i genotipi vengono analizzati in gruppi di 16 o 32 mosche.
<ol><li>Preparare un numero appropriato di tubi con cibo volante a un'estremità. NOTA: La dieta e il metabolismo sono noti per influenzare il sonno33,34, quindi è particolarmente importante posizionare le mosche sullo stesso cibo su cui sono state allevate.</li>
	<li>Sigillare l'estremità dei tubi con cera. NOTA: la privazione del sonno e il rimbalzo sono un esperimento di cinque giorni e il cibo può asciugarsi se non adeguatamente sigillato. In tubi adeguatamente sigillati, il cibo può essere mantenuto per 10 giorni o più. Pertanto, è fondamentale assicurarsi che le estremità dei tubi siano ben sigillate. Tuttavia, le mosche possono anche rimanere attaccate al cibo umido. Pertanto, aiuta a realizzare tubi 1-2 giorni prima dell'inizio dell'esperimento.</li>
	<li>Posizionare individualmente la scia, comportandosi mosche in tubi di vetro lunghi 65 mm per la registrazione del sonno utilizzando un aspiratore e collegare l'estremità dei tubi con un tappo di schiuma. NOTA: le mosche non vengono mai riconseposte all'anestesia co2 quando inseriscono le mosche in tubi per la registrazione del sonno. L'aspiratore è costituito da tubi di gomma con un'estremità ricoperta di garza e inserita in una punta della pipetta da 1 mL.</li>
</ol>3. Registrazione del sonno
<ol><li>Carica le mosche in tubi nei monitor di attività per monitorare il sonno. NOTA: le rocce SNAP monitorano avanti e indietro da -60° a +60° ogni ~10 s. I monitor sono tenuti a -60 ° per ~ 5.9s ; ci vogliono ~ 2,9 s perché il vassoio che tiene i monitor passi da -60 ° a +60 ° e ~ 1 s per tornare indietro da +60 ° a -60 °. La lunghezza del ciclo può essere modificata secondo necessità regolando la tensione fornita al motore.<ol>
<li>Fare attenzione a garantire che i tubi siano posizionati nei monitor di attività con l'orientamento corretto. Nell'orientamento corretto, l'estremità del tubo con il cibo è nella parte superiore dello SNAP per garantire che le mosche non vengano spinte nel cibo. Inoltre, la fine con il cibo è sul lato del monitor con il jack di registrazione del sonno. Ciò consente ai monitor di attività di essere orientati correttamente nello SNAP per un'efficiente privazione del sonno e contemporaneamente monitorare l'attività.</li>
	</ol>
</li>
	<li>Posizionare i monitor di attività nella camera di registrazione per monitorare il sonno.</li>
	<li>Monitorare il sonno per almeno due giorni interi per stimare il sonno basale. NOTA: il giorno in cui le mosche vengono caricate nei monitor di attività è in genere escluso come giorno di adattamento per consentire alle mosche di adattarsi ad essere alloggiate in tubi. Il sonno di base viene registrato per almeno due giorni interi (48 ore) a partire dalla mattina successiva al giorno in cui vengono caricate le mosche.</li>
	<li>Salva il conteggio dell'attività locomotoria delle mosche nei contenitori di 1 minuto dal momento delle luci di un determinato giorno alle luci del giorno precedente utilizzando il software di registrazione delle attività (ad esempio, dalle 8:00 alle 8:00).</li>
	<li>Stimare il sonno dai dati dell'attività locomotoria con macro personalizzate utilizzando 5 minuti di inattività come soglia per un attacco di sonno35. NOTA: un certo numero di metriche del sonno vengono calcolate dai conteggi dell'attività locomotoria. Questi includono il sonno in min / h oltre 24 ore, il tempo di sonno totale in 24 ore, la durata media e massima del sonno diurno e notturno36.</li>
</ol>4. Privazione del sonno e recupero
<ol><li>Poiché le mosche possono essere private del sonno per periodi di tempo variabili (ad esempio, 12 h, 24 h e 36 h) e il sonno di recupero può anche essere valutato a vari intervalli (ad esempio, 6 h, 12 h, 24 h e 48 h), determinare la durata del recupero per necessità sperimentale. Il recupero del sonno può essere visualizzato utilizzando un grafico di guadagno / perdita di sonno o esaminando la percentuale di sonno recuperato in un intervallo predeterminato (ad esempio, 6 ore).</li>
	<li>Se il sonno è stabile nei due giorni di base, il terzo giorno, inserire i monitor di attività nello SNAP per la privazione del sonno durante la notte. NOTA: le mosche mostreranno un robusto rimbalzo del sonno su un intervallo di tempi di sonno8,32,37,38,ma il sonno deve essere stabile per valutare in modo affidabile il sonno di rimbalzo. Il sonno è stabile quando la differenza di sonno tra i giorni di base è di ± 100 minuti.</li>
	<li>Assicurarsi che i monitor di attività siano fissati in posizione con i pin del supporto del monitor, i cavi del monitor collegati e i monitor orientati correttamente con l'estremità con il cibo sul retro e le barriere di plastica davanti (Figura 1). NOTA: SNAP è progettato in modo che la camma ruoti una volta ogni 10 s (Figura 1). L'inserto in plastica ripristina i tubi spingendo i tubi indietro quando l'apparecchio è in posizione "up". Il ripristino dei tubi è importante per garantire che tutti i tubi abbiano l'intera gamma di movimento all'inizio di ogni ciclo.</li>
	<li>Scollegare i monitor di attività e togliere i monitor dallo SNAP immediatamente dopo l'accendersi dopo la privazione del sonno durante la notte. NOTA: è fondamentale che la privazione del sonno sia terminata e che le mosche siano poste in recupero immediatamente dopo le luci accese dopo 12 ore di privazione del sonno durante la notte. Anche un ritardo di 20-30 minuti nel mettere le mosche in recupero può interferire con l'estensione del sonno di rimbalzo.</li>
	<li>Posiziona le mosche in una camera di registrazione dove saranno indisturbate per due giorni (48 ore) per monitorare il sonno di recupero. NOTA: Se la camera di registrazione viene utilizzata per altri esperimenti, è necessario prestare particolare attenzione per evitare di stimolare il recupero delle mosche.</li>
	<li>Calcola la quantità di sonno perso. Per ogni singola mosca, calcolare la differenza oraria tra il sonno ottenuto durante la privazione del sonno e l'ora corrispondente durante il basale; sommare le differenze orarie per calcolare il sonno totale perso.</li>
	<li>Calcola la quantità di sonno recuperato. Per ogni singola mosca, calcolare la differenza oraria tra il sonno ottenuto durante il recupero e l'ora corrispondente durante il basale; sommare le differenze orarie per calcolare il sonno totale guadagnato. NOTA: Se una mosca è effettivamente privata del sonno è empirico. Pertanto, lo sperimentatore dovrebbe esaminare la percentuale di sonno perso. Se la mosca non ha perso una quantità sufficiente di sonno può essere esclusa dall'analisi. Anche se questo potrebbe essere richiesto per altri approcci di privazione del sonno, è raramente se mai richiesto per lo SNAP. Più comunemente, il sonno potrebbe non essere stabile in una determinata mosca prima dell'inizio della privazione del sonno. Se il sonno non è stabile, l'omeostasi non può essere calcolata. Accettiamo una differenza massima di ± 100 minuti di sonno calcolata prima dell'inizio della privazione del sonno come candidati per l'inclusione. A volte, il sonno di una singola mosca è distribuito in modo non uniforme durante il giorno di 24 ore (ad esempio, alcuni individui possono ottenere il 60-70% della loro quota di sonno durante il giorno e quindi perdere solo una piccola parte della loro quota di sonno di 24 ore quando privati per 12 ore di notte). Queste mosche possono essere valutate separatamente.</li>
	<li>Calcola la percentuale media di sonno recuperato (rispetto al basale) su 12 ore, 24 ore e 48 ore del periodo di recupero per ciascun genotipo.</li>
	<li>Dai dati del sonno, calcola la durata media e massima del sonno diurno sul basale e i giorni di recupero per ciascun genotipo. NOTA: il sonno di rimbalzo nelle mosche è caratterizzato da una maggiore quantità di sonno e da una maggiore profondità del sonno nei giorni di recupero. Il consolidamento del sonno viene utilizzato come misura della profondità del sonno. Le soglie di eccitazione potrebbero anche essere utilizzate come misura della profondità del sonno.</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=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>

Gli autori non hanno nulla da rivelare.

Il sonno in Drosophila è stato caratterizzato in modo indipendente nel 2000, da due gruppi4,5. In questi studi pionieristici, le mosche sono state private del sonno da una manipolazione delicata (cioè la privazione della mano) e hanno dimostrato di mostrare una robusta risposta omeostatica alla privazione del sonno notturno. È importante sottolineare che con qualsiasi esperimento di privazione del sonno è fondamentale controllare i potenziali effetti...

Il sonno è quasi universale negli animali, ma la sua funzione rimane poco chiara. L'omeostasi del sonno, l'aumento compensatorio del sonno dopo la privazione del sonno, è una proprietà determinante del sonno, che è stata utilizzata per caratterizzare gli stati del sonno in un certo numerodi animali1,2,3,4,5.
Il sonno nella mosca ha molte somiglianze con il sonno umano, tra cui una robusta risposta omeostatica alla perdita di sonno4,5. Numerosi studi sul sonno nella mosca hanno utilizzato la privazione del sonno sia per dedurre la funzione del sonno esaminando le conseguenze negative che derivano dalla veglia prolungata, sia per comprendere la regolazione del sonno determinando i meccanismi neurobiologici che controllano la regolazione omeostatica del sonno. Così le mosche private del sonno hanno dimostrato di mostrare menomazioni nell'apprendimento e nella memoria6,7,8,9,10,11,12, plasticità strutturale13,14,15, attenzione visiva16, recupero da lesioni neuronali17,18, accoppiamento e comportamenti aggressivi19, 20, proliferazione cellulare21e risposte allo stress ossidativo22,23 per citarne alcuni. Inoltre, leindagini sui meccanismi neurobiologici che controllano il sonno di rimbalzo hanno prodotto informazioni critiche sul meccanismo neuronale che costituisce l'omeostato del sonno8,9,23, 24,25,26,27,28,29 . Infine, oltre a rivelare intuizioni fondamentali sulla funzione del sonno negli animali sani, gli studi sulla privazione del sonno hanno anche informato le intuizioni sulla funzione del sonno negli statipatologici 30,31.
Mentre la privazione del sonno è innegabilmente uno strumento potente, con qualsiasi esperimento di privazione del sonno, è importante distinguere i fenotipi che derivano dalla veglia prolungata, da quelli indotti dallo stimolo utilizzato per mantenere sveglio l'animale. La privazione del sonno per privazione della mano o manipolazione delicata, è generalmente considerata come lo standard per la privazione del sonno minimamente dirompente. Qui descriviamo un protocollo per privare il sonno delle mosche usando il Sleep Nullifying Apparatus (SNAP). Lo SNAP è un dispositivo che fornisce uno stimolo meccanico alle mosche ogni 10 secondi, mantenendo sveglie le mosche inducendo geotaxi negativi (Figura 1). Lo SNAP priva efficacemente le mosche di >98% del sonno notturno, anche nelle mosche con un elevato livello di sonno8,32. Lo SNAP è stato calibrato su mosche sensibili al botto, l'agitazione delle mosche nello SNAP non danneggia le mosche; la privazione del sonno con lo SNAP induce un rimbalzo paragonabile a quello ottenuto dalla privazione della mano7. Lo SNAP è quindi un metodo robusto per privare le mosche del sonno mentre controlla gli effetti dello stimolo eccitante.

<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

L'omeostasi del sonno, l'aumento del sonno osservato dopo la perdita di sonno, è uno dei criteri di definizione utilizzati per identificare il sonno in tutto il regno animale. Di conseguenza, la privazione del sonno e la restrizione del sonno sono strumenti potenti che sono comunemente usati per fornire informazioni sulla funzione del sonno. Tuttavia, gli esperimenti di privazione del sonno sono intrinsecamente problematici in quanto lo stimolo di privazione stesso può essere la causa dei cambiamenti osservati nella fisiologia e nel comportamento. Di conseguenza, le tecniche di privazione del sonno di successo dovrebbero mantenere gli animali svegli e, idealmente, provocare un robusto rimbalzo del sonno senza indurre anche un gran numero di conseguenze indesiderate. Qui, descriviamo una tecnica di privazione del sonno per Drosophila melanogaster. Lo Sleep Nullifying Apparatus (SNAP) somministra uno stimolo ogni 10 secondi per indurre geotaxi negativi. Sebbene lo stimolo sia prevedibile, lo SNAP previene efficacemente >95% del sonno notturno anche nelle mosche con un elevato calo del sonno. È importante sottolineare che la successiva risposta omeostatica è molto simile a quella ottenuta utilizzando la privazione della mano. I tempi e la spaziatura degli stimoli possono essere modificati per ridurre al minimo la perdita di sonno e quindi esaminare gli effetti non specifici dello stimolo sulla fisiologia e sul comportamento. Lo SNAP può essere utilizzato anche per la restrizione del sonno e per valutare le soglie di eccitazione. Lo SNAP è una potente tecnica di interruzione del sonno che può essere utilizzata per comprendere meglio la funzione del sonno.

Il Canton S (Cs) è stato utilizzato come ceppo selvatico. Le mosche sono state mantenute su una luce di 12 ore: 12 ore di buio e sonno privato per 12 ore durante la notte. L'ispezione dei profili del sonno delle mosche Cs nel giorno di base (bs), nel giorno di privazione del sonno (sd) e in due giorni di recupero (rec1 e rec2)(Figura 2A)suggerisce che le mosche sono state effettivamente private del sonno nello SNAP e hanno recuperato il sonno durante il gior...

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

La privazione del sonno è un potente strumento per studiare la funzione e la regolazione del sonno. Descriviamo un protocollo per privare di sonno Drosophila usando l'apparato annullante del sonno e per determinare l'entità del sonno di rimbalzo indotto dalla privazione.

L'apparato che annulla il sonno: un metodo altamente efficiente per privare la drosophila del sonno

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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Neuroscience

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

3.3K Views.  Washington University School of Medicine. La privazione del sonno è un potente strumento per studiare la funzione e la regolazione del sonno. Descriviamo un protocollo per privare di sonno Drosophila usando l'apparato annullante del sonno e per determinare l'entità del sonno di rimbalzo indotto dalla privazione.

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.

Nuovo Apparato e metodo per il rinforzo della droga

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

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Neuroscienze

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.

Saggio attività locomotoria per studiare ritmi circadiani del sonno e parametri in Drosophila</em

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

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non Laser Capture Microscopy approccio per la Microdissezione delle Regioni discreti cervello di topo per la totale isolamento di RNA e valle Next-Generation Sequencing e profili di espressione genica

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.

Elettroencefalografia simultanea, in tempo reale misura della concentrazione di lattato e la manipolazione Optogenetic di attività neuronale nella corteccia cerebrale Rodentia

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.

Un metodo di nodose Ganglia iniezione 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 ...

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.

Procedura di registrazione poligrafica per la misurazione del sonno nei topi

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.

Manipolazione optogenetica dei circuiti neurali durante il monitoraggio degli stati di sonno/veglia nei topi

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.

Come ottenere potenziali visivi affidabili legati agli eventi visivi nei neonati

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

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

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Misurazione dei meccanismi neurali alla base del consolidamento della memoria dipendente dal sonno durante le sinisine nella prima infanzia

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Registrazione elettrocorticografica delle aree della corteccia cerebrale manipolate utilizzando un virus adeno-associato che prende di mira Cofilin nei topi

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

Methods Article

L'apparato che annulla il sonno: un metodo altamente efficiente per privare la drosophila del sonno