Este trabajo fue apoyado por las subvenciones de los NIH 5R01NS051305-14 y 5R01NS076980-08 a PJS.

1. Preparación experimental
<ol><li>Recolecte moscas a medida que eclosión en viales, separando las moscas macho y hembra. NOTA: Los experimentos de sueño se realizan comúnmente con moscas hembras. Es importante recolectar hembras vírgenes. Las hembras apareadas pondrán huevos que eclosionan en larvas complicando el análisis de los datos.</li>
	<li>Moscas domésticas de un solo sexo en grupos de <20. NOTA: La vivienda de moscas en un entorno socialmente enriquecido (grupos de >50) modula la unidad de sueño6,13 mediciones potencialmente confusas del sueño de rebote. Además, después del enriquecimiento social, el sueño disminuirá en unos pocos días6. Por lo tanto, el sueño basal no es estable, lo que complica el análisis del sueño de rebote. Mantener las moscas en grupos de <20 evita esta posible confusión.</li>
	<li>Mantenga las moscas en viales durante 3-5 días en un ambiente ligero y con humedad controlada. NOTA: La edad y la madurez de las moscas influyen fuertemente en el sueño. El sueño es alto en las moscas de un día de edad y se estabiliza a los 3-5 días de edad4. Las moscas se mantienen típicamente en un horario oscuro de 12 h: 12 h a un 50% de humedad.</li>
</ol>2. Preparación de tubos para la grabación del sueño
NOTA: El sueño se monitorea usando monitores de actividad locomotora. Un monitor puede contener 32 moscas alojadas individualmente en tubos de 5 mm de diámetro. Típicamente, los genotipos se analizan en grupos de 16 o 32 moscas.
<ol><li>Prepare un número apropiado de tubos con comida para moscas en un extremo. NOTA: Se sabe que la dieta y el metabolismo influyen en el sueño33,34, por lo tanto, es particularmente importante colocar moscas en el mismo alimento en el que fueron criadas.</li>
	<li>Selle el extremo de los tubos con cera. NOTA: La privación del sueño y el rebote es un experimento de cinco días, y la comida puede secarse si no se sella adecuadamente. En tubos debidamente sellados, los alimentos se pueden mantener durante 10 días o más. Por lo tanto, es fundamental asegurarse de que los extremos de los tubos estén bien sellados. Sin embargo, las moscas también pueden atascarse en la comida húmeda. Por lo tanto, ayuda a hacer tubos 1-2 días antes del inicio del experimento.</li>
	<li>Coloque individualmente las moscas de estela, que se comportan en tubos de vidrio de 65 mm de largo para la grabación del sueño con un aspirador y enchufe el extremo de los tubos con un tapón de espuma. NOTA: Las moscas nunca se vuelven a exponer a la anestesia con CO2 cuando se colocan moscas en tubos para registrar el sueño. El aspirador está hecho de tubos de goma con un extremo cubierto con gasa e insertado en una punta de pipeta de 1 ml.</li>
</ol>3. Grabación del sueño
<ol><li>Cargue moscas en tubos en monitores de actividad para monitorear el sueño. NOTA: El SNAP monitorea las rocas de ida y vuelta de -60 ° a + 60 ° cada ~ 10 s. Los monitores se mantienen a -60 ° durante ~ 5.9s; se necesitan ~ 2.9 s para que la bandeja que sostiene los monitores se mueva de -60 ° a + 60 ° y ~ 1 s para retroceder de + 60 ° a -60 °. La duración del ciclo se puede alterar según sea necesario ajustando el voltaje suministrado al motor.<ol>
<li>Tenga cuidado de asegurarse de que los tubos se coloquen en los monitores de actividad en la orientación correcta. En la orientación correcta, el extremo del tubo con comida está en la parte superior del SNAP para garantizar que las moscas no se empujen hacia la comida. Además, el extremo con la comida está en el lado del monitor con el conector de grabación del sueño. Esto permite que los monitores de actividad se orienten correctamente en el SNAP para una privación eficiente del sueño mientras se monitorea simultáneamente la actividad.</li>
	</ol>
</li>
	<li>Coloque monitores de actividad en la cámara de grabación para monitorear el sueño.</li>
	<li>Controle el sueño durante al menos dos días completos para estimar el sueño inicial. NOTA: El día en que las moscas se cargan en monitores de actividad generalmente se excluye como un día de adaptación para permitir que las moscas se adapten a ser alojadas en tubos. El sueño basal se registra durante al menos dos días completos (48 horas) a partir de la mañana siguiente al día en que se cargan las moscas.</li>
	<li>Guarde los recuentos de actividad locomotora de las moscas en contenedores de 1 minuto desde el momento de las luces en un día determinado hasta las luces del día anterior utilizando un software de registro de actividad (por ejemplo, de 8 AM a 8 AM).</li>
	<li>Calcule el sueño a partir de los datos de actividad locomotora con macros personalizadas utilizando 5 minutos de inactividad como umbral para un episodio de sueño35. NOTA: Una serie de métricas de sueño se calculan a partir de los recuentos de actividad locomotora. Estos incluyen el sueño en min/h durante 24 h, el tiempo total de sueño en 24 h, la duración media y máxima del sueño diurno y nocturno36.</li>
</ol>4. Privación del sueño y recuperación
<ol><li>Como las moscas pueden estar privadas de sueño durante períodos de tiempo variables (por ejemplo, 12 h, 24 h y 36 h) y el sueño de recuperación también se puede evaluar a varios intervalos (por ejemplo, 6 h, 12 h, 24 h y 48 h), determine la duración de la recuperación por necesidad experimental. La recuperación del sueño se puede visualizar utilizando una gráfica de ganancia/pérdida de sueño o examinando el porcentaje de sueño recuperado durante un intervalo predeterminado (por ejemplo, 6 h).</li>
	<li>Si el sueño es estable durante los dos días de referencia, en el tercer día, coloque monitores de actividad en el SNAP para la privación del sueño durante la noche. NOTA: Las moscas exhibirán un rebote de sueño robusto en un rango de tiempos de sueño8,32,37,38,pero el sueño debe ser estable para evaluar de manera confiable el sueño de rebote. El sueño es estable cuando la diferencia en el sueño entre los días basales es de ± 100 min.</li>
	<li>Asegúrese de que los monitores de actividad estén asegurados en su lugar con pines de soporte para monitores, cables de monitor enchufados y monitores orientados correctamente con el extremo con alimentos en la parte posterior y barreras de plástico en la parte delantera(Figura 1). NOTA: El SNAP está diseñado para que la leva gire una vez cada 10 s (Figura 1). El inserto de plástico restablece los tubos empujando los tubos hacia atrás cuando el aparato está en la posición "hacia arriba". El restablecimiento de los tubos es importante para garantizar que todos los tubos tengan el rango completo de movimiento al comienzo de cada ciclo.</li>
	<li>Desconecte los monitores de actividad y saque los monitores del SNAP inmediatamente después de encender las luces después de la privación del sueño durante la noche. NOTA: Es fundamental que se ponga fin a la privación del sueño, y las moscas se colocan en recuperación inmediatamente después de las luces encendidas después de 12 h de privación del sueño durante la noche. Incluso un retraso de 20-30 minutos en la colocación de moscas en la recuperación puede interferir con la extensión del sueño de rebote.</li>
	<li>Coloque las moscas en una cámara de grabación donde no serán molestadas durante dos días (48 h) para monitorear el sueño de recuperación. NOTA: Si la cámara de grabación se está utilizando para otros experimentos, se debe tener especial cuidado para evitar estimular las moscas en recuperación.</li>
	<li>Calcule la cantidad de sueño perdido. Para cada mosca individual, calcule la diferencia horaria entre el sueño obtenido durante la privación del sueño y la hora correspondiente durante la línea de base; sumar las diferencias horarias para calcular el sueño total perdido.</li>
	<li>Calcula la cantidad de sueño recuperado. Para cada mosca individual, calcule la diferencia horaria entre el sueño obtenido durante la recuperación y la hora correspondiente durante la línea de base; sumar las diferencias horarias para calcular el sueño total ganado. NOTA: Si una mosca está realmente privada de sueño es empírico. Por lo tanto, el experimentador debe examinar el porcentaje de sueño perdido. Si la mosca no ha perdido una cantidad suficiente de sueño, puede excluirse del análisis. Aunque esto podría ser necesario para otros enfoques de privación del sueño, rara vez o nunca se requiere para el SNAP. Más comúnmente, el sueño puede no ser estable en una mosca dada antes del inicio de la privación del sueño. Si el sueño no es estable, no se puede calcular la homeostasis. Aceptamos una diferencia máxima de ± 100 min de sueño calculada antes del inicio de la privación del sueño como candidatos para la inclusión. En ocasiones, el sueño de una mosca individual se distribuye de manera desigual a lo largo del día de 24 horas (por ejemplo, algunos individuos pueden obtener el 60-70% de su cuota de sueño durante el día y, por lo tanto, solo pierden una pequeña proporción de su cuota de sueño de 24 horas cuando se les priva durante 12 horas por la noche). Estas moscas se pueden evaluar por separado.</li>
	<li>Calcular el porcentaje medio de sueño recuperado (en relación con el valor basal) durante 12 h, 24 h y 48 h del período de recuperación para cada genotipo.</li>
	<li>A partir de los datos de sueño, calcule la duración promedio y máxima del sueño diurno en la línea de base y los días de recuperación para cada genotipo. NOTA: El sueño de rebote en las moscas se caracteriza por un aumento de la cantidad de sueño y una mayor profundidad del sueño en los días de recuperación. La consolidación del sueño se utiliza como medida de la profundidad del sueño. Los umbrales de excitación también podrían usarse como una medida de la profundidad del sueño.</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>

Los autores no tienen nada que revelar.

El sueño en Drosophila se caracterizó de forma independiente en 2000, por dos grupos4,5. En estos estudios pioneros, las moscas se vieron privadas del sueño por un manejo suave (es decir, privación de la mano) y se demostró que exhiben una respuesta homeostática robusta a la privación del sueño durante la noche. Es importante destacar que con cualquier experimento de privación del sueño es crucial controlar los posibles efectos de confusión de...

El sueño es casi universal en los animales, sin embargo, su función sigue sin estar clara. La homeostasis del sueño, el aumento compensatorio del sueño después de la privación del sueño, es una propiedad definitoria del sueño, que se ha utilizado para caracterizar los estados de sueño en un número de animales1,2,3,4,5.
El sueño en la mosca tiene muchas similitudes con el sueño humano, incluida una respuesta homeostática robusta a la pérdida de sueño4,5. Numerosos estudios sobre el sueño en la mosca han utilizado la privación del sueño tanto para inferir la función del sueño al examinar las consecuencias adversas que se derivan de la vigilia prolongada, como para comprender la regulación del sueño mediante la determinación de los mecanismos neurobiológicos que controlan la regulación homeostática del sueño. Así, se demostró que las moscas privadas de sueño exhiben deficiencias en el aprendizaje y la memoria6,7,8,9,10,11, 12,plasticidad estructural13,14,15,atención visual16,recuperación de una lesión neuronal17,18,apareamiento y comportamientos agresivos19, 20, proliferación celular21, y respuestas al estrés oxidativo22,23 por nombrar algunos. Además, las investigaciones sobre los mecanismos neurobiológicos que controlan el sueño de rebote han arrojado información crítica sobre la maquinaria neuronal que constituye el homeostato del sueño8,9,23,24,25,26,27,28,29 . Finalmente, además de revelar información fundamental sobre la función del sueño en animales sanos, los estudios de privación del sueño también han informado sobre la función del sueño en estados enfermos30,31.
Si bien la privación del sueño es innegablemente una herramienta poderosa, con cualquier experimento de privación del sueño, es importante distinguir los fenotipos que resultan de la vigilia prolongada, de los inducidos por el estímulo utilizado para mantener al animal despierto. La privación del sueño por privación de la mano o manejo suave, generalmente se considera que establece el estándar para la privación del sueño mínimamente perturbadora. Aquí describimos un protocolo para privar del sueño a las moscas utilizando el Aparato de Anulación del Sueño (SNAP). El SNAP es un dispositivo que entrega un estímulo mecánico a las moscas cada 10 s, manteniendo a las moscas despiertas mediante la inducción de geotaxis negativas(Figura 1). El SNAP priva eficientemente a las moscas del >98% del sueño nocturno, incluso en moscas con alto nivel de sueño8,32. El SNAP ha sido calibrado en moscas sensibles a la explosión, la agitación de las moscas en el SNAP no daña a las moscas; la privación del sueño con el SNAP induce un rebote comparable al obtenido por la privación de la mano7. El SNAP es, por lo tanto, un método robusto para privar del sueño a las moscas mientras controla los efectos del estímulo excitante.

<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

La homeostasis del sueño, el aumento del sueño observado después de la pérdida de sueño, es uno de los criterios definitorios utilizados para identificar el sueño en todo el reino animal. Como consecuencia, la privación del sueño y la restricción del sueño son herramientas poderosas que se utilizan comúnmente para proporcionar información sobre la función del sueño. Sin embargo, los experimentos de privación del sueño son inherentemente problemáticos en el momento en que el estímulo de privación en sí mismo puede ser la causa de los cambios observados en la fisiología y el comportamiento. En consecuencia, las técnicas exitosas de privación del sueño deben mantener a los animales despiertos e, idealmente, dar como resultado un rebote robusto del sueño sin inducir también una gran cantidad de consecuencias no deseadas. Aquí, describimos una técnica de privación del sueño para Drosophila melanogaster. El Aparato Anulador del Sueño (SNAP) administra un estímulo cada 10s para inducir geotaxis negativas. Aunque el estímulo es predecible, el SNAP previene eficazmente el sueño >95% de la noche, incluso en moscas con un alto impulso de sueño. Es importante destacar que la respuesta homeostática posterior es muy similar a la lograda mediante la privación de la mano. El tiempo y el espaciamiento de los estímulos se pueden modificar para minimizar la pérdida de sueño y, por lo tanto, examinar los efectos no específicos del estímulo en la fisiología y el comportamiento. El SNAP también se puede utilizar para la restricción del sueño y para evaluar los umbrales de excitación. El SNAP es una poderosa técnica de interrupción del sueño que se puede utilizar para comprender mejor la función del sueño.

Canton S (Cs) se utilizó como una cepa de tipo salvaje. Las moscas se mantuvieron en un horario de luz de 12 h: 12 h de oscuridad y se privó de sueño durante 12 horas durante la noche. La inspección de los perfiles de sueño de las moscas Cs en el día basal (bs), el día de privación del sueño (sd) y dos días de recuperación (rec1 y rec2)(Figura 2A)sugiere que las moscas fueron efectivamente privadas de sueño en el SNAP, y recuperaron el sueño dura...

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 privación del sueño es una herramienta poderosa para investigar la función y regulación del sueño. Describimos un protocolo para privar del sueño a Drosophila utilizando el Aparato Anulador del Sueño, y para determinar el alcance del sueño de rebote inducido por la privación.

El aparato anulador del sueño: un método altamente eficiente de privación del sueño a Drosophila

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 vistas.  Washington University School of Medicine. La privación del sueño es una herramienta poderosa para investigar la función y regulación del sueño. Describimos un protocolo para privar del sueño a Drosophila utilizando el Aparato Anulador del Sueño, y para determinar el alcance del sueño de rebote inducido por la privación.

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.

Novela Aparato y método para el fortalecimiento de Drogas

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

Investigación

Neurociencias

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.

Ensayo Actividad locomotora para el Estudio de los ritmos circadianos y del sueño en los parámetros 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.

La criopreservación de tejido cortical Bloques para la generación de cultivos neuronales altamente enriquecido

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.

Sin láser y de captura de enfoque de Microscopía de la microdisección de regiones discretas del cerebro del ratón para el aislamiento de ARN total y Downstream secuenciación de próxima generación y de perfiles de expresión génica

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.

Electroencefalografía simultánea, en tiempo real Medición de la concentración de lactato y Manipulación optogenético de la actividad neuronal en la corteza cerebral de roedores

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 método de nodose Ganglios Inyección en rata Sprague-Dawley

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.

Un método para infligir Cerrado Jefe lesión cerebral traumática en<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.

Poligráfico Procedimiento de Grabación en medición del sueño en ratones

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.

Manipulación optogenética de circuitos neuronales durante la supervisión de los estados de sueño/vigilia en ratones

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.

El aparato anulador del sueño: un método altamente eficiente de privación del sueño a Drosophila

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