The authors have nothing to disclose.

Todos los procedimientos y pruebas de comportamiento fueron aprobados por la Junta de Ética de Experimentación Animal de la Universidad de MacEwan con el número de protocolo 11/06/12, que está en cumplimiento con el Consejo Canadiense para las directrices del Cuidado de Animales para el cuidado y uso de animales de experimentación. 1. Preparar tanques de dosificación, soluciones y Horario Administración <ol><li> Preparar un programa de administración de tal manera que los animales se dosifican en el mismo entorno y durante la misma hora del día para evitar cualquier confunde de tiempo o de empuje a los estímulos visuales. </li><li> Obtener la mayor cantidad, 1,5 L, tanques de desove polipropileno claras idénticas como sea necesario para el número de tamaños de los grupos. Use grupos de 8 peces por tanque, lo que permite 2 grupos de peces a ensayar por día más tarde en el procedimiento (ver paso 3). Utilice un tanque de retención y un tanque de dosificación por grupo (2x número total de grupos). <ol><li> Coloque 400 micras inserciones de desove en todos los tanques de almacenamiento. Fenfermos los tanques con agua de hábitat o agua de ósmosis inversa a la temperatura correcta (para el pez cebra, 25-28 ° C) que sea consistente con los peces de temperatura son normalmente alojados en. NOTA: No puede haber interacciones químicas no deseadas entre algunos medicamentos y los componentes químicos del agua tamponada hábitat. En esta situación, utilizar agua de ósmosis inversa tamponada con sales mínimas o ninguna acuario para la administración de fármacos, así como para los grupos de control. </li><li> Asegúrese de que los tanques se encuentran en un ambiente neutral para evitar el pescado acondicionado a los estímulos visuales externos durante la dosificación. </li></ol></li><li> Preparar la solución de fármaco. Mezcle la cantidad apropiada de la droga con agua hábitat en los tanques de desove. Preparar la solución de etanol al 0,2% mediante la combinación de 3 ml de un etanol de alto grado (95% de etanol no desnaturalizado) con 1497 ml de agua. Preparar la solución de etanol al 1,4% mediante la combinación de 21 ml de etanol con 1479 ml de agua. </li></ol> 2. Compensación de Pesca y EtProcedimiento Administración Hanol <ol><li> Pescado con cuidado neto de sus tanques de hábitat y la transferencia en el tanque de retención adecuado que contiene el inserto de desove. Lo ideal sería que albergar a los peces en el inserto de desove para eliminar la compensación total. </li><li> Con todos los peces en sus respectivos tanques de almacenamiento, levante suavemente el desove insertar fuera del tanque de retención y colocarlo en el depósito de solución de fármaco apropiada (Figura 1A). <ol><li> Registre el tiempo de dosificación según sea necesario. Utilice 30 min en la solución de etanol para el procedimiento descrito aquí. </li><li> Si es posible, tener asistentes ayudan con la transferencia de todos los grupos experimentales para la solución de fármaco simultáneamente para garantizar el tiempo de dosificación precisa. Por otra parte, la transferencia de un grupo a la vez y hacer un seguimiento de la dosificación veces (Figura 1A) grupos individuales. </li></ol></li><li> Al final del período de dosificación requerida, quitar el pescado de la solución de etanol levantando cuidadosamente el inserto de desovede la solución de fármaco y colocando suavemente de nuevo en el tanque de retención. </li><li> Suavemente neta a los peces en los tanques de almacenamiento y colocarlos de nuevo en su hábitat hasta la próxima vez que la dosificación programada, o coloque el desove insertar de nuevo en el tanque de retención para eliminar la compensación. </li><li> Como se mencionó anteriormente, si es posible dentro de los parámetros de equipo de alojamiento de los animales, la casa de los animales en el mismo tanque y el inserto de desove que se utiliza como el tanque de retención. Esto eliminará la compensación por completo durante el proceso de administración. </li></ol> 3. Pruebas de Comportamiento <ol><li> Obtener una luz / arena oscuro 9,5 cm de ancho por 55 cm de largo y 9,5 cm de profundidad con un piso impermeable blanco (Figura 1B). Coloque papel no reflectante impermeable blanco y negro a las paredes interiores de la arena usando velcro, con la mitad de la arena cubierta en blanco y negro en medio cubierto. Llenar la arena hasta una profundidad de 5 cm con agua hábitat a una temperatura de 25-28 ° C. Maintain esta temperatura durante toda la prueba. </li><li> Minimalize estímulos visuales externas mediante la construcción de un recinto blanco de tres lados de la arena que se encuentra en. Asegúrese de que el área de pruebas tiene difusa iluminación cenital que no causa reflejos en la superficie del agua, sin embargo, es lo suficientemente brillante para el software de seguimiento de movimiento, o post cuantificación manual de -hoc a partir de imágenes de vídeo. </li><li> Coloque la arena en el recinto y configurar los parámetros de grabación y análisis de movimiento del software de seguimiento de la conducta. Establezca la duración del ensayo a 5-15 minutos, dependiendo de la pregunta de investigación. NOTA: En este caso, hemos utilizado 5 min. </li><li> Transportar el grupo de peces a ensayar para el área de investigación en el tanque de hábitat y colocarlos fuera del recinto de la arena. Aclimatar a los peces durante 10 minutos. </li><li> Un pez con cuidado neto del grupo y el lugar adecuado en el centro de la luz / arena oscura, asegurándose de liberar a los peces cuando se coloca paralela al eje largo de la arena para evIdentificación del sesgar el pescado a la luz o de la zona oscura. </li><li> Comience comportamiento a grabar inmediatamente después de soltar al animal. Esté atento a cualquier problema de software con el seguimiento de los peces o de peces saltando o congelación. Gire la arena 180 ° después de la mitad de los sujetos se ha probado para prevenir cualquier confunde debido a los sesgos resultantes de qué extremo de la arena está orientada hacia el extremo abierto de la caja. </li><li> Después de que el juicio ha terminado, suavemente red y eliminar el pescado de la arena a un tanque de almacenamiento o depósito de hábitat. </li></ol> 4. Análisis <ol><li> Examine el tiempo dedicado a la luz frente a las zonas oscuras. Para cada grupo y cada uno de los peces, obtenga el tiempo relativo transcurrido en las zonas claras y oscuras y analizar el uso de una muestra de t-test (o prueba de Wilcoxon para datos no paramétricos; diferencia de (la mitad del tiempo total de prueba) 150 seg) para determinar si los grupos prefieren significativamente un área sobre el otro. </li><li> Para comparar las preferencias, calcular una prefeíndice rencia restando el tiempo pasado en la zona de luz desde el tiempo pasado en la zona oscura y comparar las diferencias entre grupos. pruebas t- se puede utilizar para comparar dos grupos. Comparación de varios grupos con un solo sentido el análisis de varianza utilizando el test post hoc de Tukey HSD cuando sea necesario (o prueba de Kruskal-Wallis con comparaciones múltiples de Dunn test post hoc de los datos no paramétricos). </li><li> Comparar la velocidad, número de transiciones de zona, serpenteantes, y la inmovilidad en todos los grupos. Utilice solo sentido el análisis de varianza utilizando el test post hoc de Tukey HSD cuando sea necesario (o prueba de Kruskal-Wallis con comparaciones múltiples de Dunn test post hoc de los datos no paramétricos). </li></ol>

<ol>
	<li>Spence, R., Gerlach, G., Lawrence, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+ecology+of+the+zebrafish,+Danio+rerio.">The behaviour and ecology of the zebrafish, Danio rerio.</a> Biological Reviews. 83 (1), 13-34 (2008).</li><li>Langheinrich, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+new+model+on+the+pharmaceutical+catwalk.">Zebrafish: A new model on the pharmaceutical catwalk.</a> BioEssays. 25 (9), 904-912 (2003).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122 (7), 2337-2343 (2012).</li><li>Miller, N., Greene, K., Dydinski, A., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nicotine+and+alcohol+on+zebrafish+(+Danio+rerio)+shoaling.">Effects of nicotine and alcohol on zebrafish ( Danio rerio) shoaling.</a> Behavioural brain research. , (2012).</li><li>Bencan, Z., Sledge, D., Levin, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buspirone,+chlordiazepoxide+and+diazepam+effects+in+a+zebrafish+model+of+anxiety.">Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety.</a> Pharmacology Biochemistry and Behavior. 94 (1), 75-80 (2009).</li><li>Holcombe, A., Howorko, A., Powell, R. A., Schalomon, M., Hamilton, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversed+Scototaxis+during+Withdrawal+after+Daily-Moderate,+but+Not+Weekly-Binge+Administration+of+Ethanol+in+Zebrafish.">Reversed Scototaxis during Withdrawal after Daily-Moderate, but Not Weekly-Binge Administration of Ethanol in Zebrafish.</a> PLoS ONE. 8 (5), (2013).</li><li>Mathur, P., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+of+acute+versus+chronic+ethanol+exposure+on+anxiety-like+behavioral+responses+in+zebrafish.">Differences of acute versus chronic ethanol exposure on anxiety-like behavioral responses in zebrafish.</a> Behavioural Brain Research. 219 (2), 234-239 (2011).</li><li>Dlugos, C., Rabin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+effects+on+three+strains+of+zebrafish:+model+system+for+genetic+investigations.">Ethanol effects on three strains of zebrafish: model system for genetic investigations.</a> Pharmacology Biochemistry and Behavior. , (2003).</li><li>Maximino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scototaxis+as+anxiety-like+behavior+in+fish.">Scototaxis as anxiety-like behavior in fish.</a> Nature Protocols. 5 (2), 209-216 (2010).</li><li>Gerlai, R., Lee, V., Blaser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+and+chronic+ethanol+exposure+on+the+behavior+of+adult+zebrafish+(Danio+rerio).">Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio).</a> Pharmacology Biochemistry and Behavior. 85 (4), 752-761 (2006).</li><li>Egan, R. J., 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=Understanding+behavioral+and+physiological+phenotypes+of+stress+and+anxiety+in+zebrafish.">Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.</a> Behavioural brain research. 205 (1), 38-44 (2009).</li><li>Gebauer, D. 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=Effects+of+anxiolytics+in+zebrafish:+Similarities+and+differences+between+benzodiazepines,+buspirone+and+ethanol.">Effects of anxiolytics in zebrafish: Similarities and differences between benzodiazepines, buspirone and ethanol.</a> Pharmacology Biochemistry and Behavior. 99 (3), 480-486 (2011).</li><li>Serra, M. C., Mattioli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+preference+of+zebrafish+(Danio+rerio)+for+a+dark+environment.">Natural preference of zebrafish (Danio rerio) for a dark environment.</a> Braz J Med Biol Res. 32 (12), 1551-1553 (1999).</li><li>Maximino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+neurochemical+changes+in+the+zebrafish+leopard+strain.">Behavioral and neurochemical changes in the zebrafish leopard strain.</a> Genes Brain Behav. 12 (5), 576-582 (2013).</li><li>Levin, E. D., Bencan, Z., Cerutti, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiolytic+effects+of+nicotine+in+zebrafish.">Anxiolytic effects of nicotine in zebrafish.</a> Physiology &amp; Behavior. 90 (1), 54-58 (2007).</li><li>Gerlai, R., Chatterjee, D., Pereira, T., Sawashima, T., Krishnannair, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+alcohol+dose:+population+differences+in+behavior+and+neurochemistry+of+zebrafish.">Acute and chronic alcohol dose: population differences in behavior and neurochemistry of zebrafish.</a> Genes, Brain and Behavior. 8 (6), 586-599 (2009).</li><li>Mathur, P., Berberoglu, M. A., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+ethanol+in+zebrafish+following+a+single+exposure.">Preference for ethanol in zebrafish following a single exposure.</a> Behavioural Brain Research. 217 (1), 128-133 (2011).</li><li>Renninger, S. 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=Investigating+the+genetics+of+visual+processing,+function+and+behaviour+in+zebrafish.">Investigating the genetics of visual processing, function and behaviour in zebrafish.</a> Neurogenetics. 12, 97-116 (2011).</li><li>Crawshaw, L. I., 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=Tolerance+and+withdrawal+in+goldfish+exposed+to+ethanol.">Tolerance and withdrawal in goldfish exposed to ethanol.</a> Physiology &amp; Behaviour. 87 (3), 460-468 (2006).</li><li>Hamilton, T. J., Holcombe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tresguerres,+M.CO2-induced+ocean+acidification+increases+anxiety+in+rockfish+via+alteration+of+GABAA+receptor+functioning.">Tresguerres, M.CO2-induced ocean acidification increases anxiety in rockfish via alteration of GABAA receptor functioning.</a> Proceedings of the Royal Society B. , (2014).</li><li>Ramsay, J. M., Feist, G. W., Varga, Z. M., Westerfield, M., Kent, M. L., Schreck, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+cortisol+response+of+zebrafish+to+acute+net+handling+stress.">Whole-body cortisol response of zebrafish to acute net handling stress.</a> Aquaculture. 297 (1-4), 157-164 (2009).</li><li>Hamilton, T. J., Paz-Yepes, J., Morrison, R. A., Palenik, B., Tresguerres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+bloom-like+concentrations+of+two+marine+Synechococcus+cyanobacteria+(strains+CC9311+and+CC9902)+differentially+alters+fish+behaviour.">Exposure to bloom-like concentrations of two marine Synechococcus cyanobacteria (strains CC9311 and CC9902) differentially alters fish behaviour.</a> Conservation Physiology. 2 (1), (2014).</li></ol>

Los autores reconocen Josué Gallup para el uso de su equipo de fotografía utilizada para la Figura 1. Este trabajo fue apoyado por una subvención de Ingeniería de Investigación (NSERC) Canadá Descubrimiento Ciencias Naturales y (a TJH).

Estudios previos que implican la administración de fármacos en el pez cebra, simplemente se han basado en la compensación de pescado para transportarlos desde su tanque de casa en la solución de fármaco 12,16. Compensación no siempre es consistente y, a menudo toma más tiempo de lo esperado debido a la respuesta de escape del pez cebra, que tiene importante variabilidad individual. Métodos de transferencia tradicionales, aunque útiles, pueden mejorarse por la disminución de la cantidad de tiempo tot...

El pez cebra (Danio rerio) es una pequeña especie de teleósteos originarios de la India, que es un organismo modelo útil para conductual 1 y 2,3 médicos de investigación. El pez cebra también se utilizan comúnmente en el ensayo de diversas sustancias farmacológicas con el fin de caracterizar su impacto en el comportamiento. Varias dosis y programas de administración de drogas se han utilizado para investigar el comportamiento del pez cebra después de la administración de compuestos tales como estimulantes, ansiolíticos 4 5 y etanol 6-8. Nuestro laboratorio ha investigado los efectos de los diferentes programas de administración de etanol sobre la ansiedad pez cebra y la locomoción en la luz / oscuridad ensayo bien validado 9, 20, también se conoce comúnmente como el ensayo scototaxic. Un nuevo método de administración de etanol fue desarrollado para aumentar la eficiencia para la administración repetitiva, diariamente durante un largo período de tiempo (21 días) 6 </sarriba&gt;. Métodos utilizados anteriormente eran prácticos, sin embargo, hemos tratado de desarrollar un método que reduce la compensación, con sus costos de tiempo asociados, y permite la administración simultánea, precisamente cronometrado de la droga de interés para un gran número de peces. En la investigación usando etanol tradicional, el pez cebra se anotó y se transfiere desde un tanque a otro que contiene la mezcla apropiada de etanol y agua 10-12. Si bien este método es ampliamente aceptada, redes pez cebra puede aumentar la variabilidad en el tiempo necesario para introducir y eliminar el pescado de la solución de fármaco. Por lo tanto, la exposición exacta para el compuesto de interés puede variar en el transcurso de un experimento con dosificación repetida. Un método que reduce las fuentes de error generados por la variabilidad en los tiempos de transporte es por lo tanto deseable. Con nuestro método somos capaces de mover todos los peces de forma simultánea, lo que resulta en el tiempo de dosificación idéntica en cada pez. Después de la exposición de etanol (que se describe aquí), el pez cebra se puede probar en cualquier number de ensayos de comportamiento, entre ellos los que evalúan la ansiedad. Dosificación grupos de peces utilizando el nuevo método tiene usos prácticos más allá de la capacidad de replicar y estandarizar la dosificación entre sujetos y entre los grupos de peces con precisión. La llegada de un nuevo software que permite el seguimiento de los peces múltiple a la vez puede ver investigadores utilizan nuestros métodos para garantizar la replicabilidad y la precisión en sus experimentos. Teniendo en cuenta el amplio uso del pez cebra como organismo modelo para la neurociencia conductual, este método aumentará la eficiencia y la practicidad en el futuro los estudios farmacológicos. En el actual paradigma, un programa de dosificación repetida fue empleado que refleja aproximadamente horarios beber humanos. Los peces fueron asignados aleatoriamente a uno de tres grupos: control, diario-moderada, o semanal-compulsivos. El horario de dosis fue de 21 días de duración, elegido porque superó significativamente los tiempos de exposición en estudios previos 7. Peces de control recibió cero alcohol, pescado al día de moderada recibió 0,2% de alcohol una vez por día, y el pescado semanal-atracón recibió 1,4% de alcohol una vez por semana. La tarea de luz / oscuridad se utilizó para evaluar la ansiedad después de 2 días de abstinencia. Esta es una prueba relativamente simple de administrar que utiliza una arena rectangular en el que las paredes en un lado son de color blanco y en el otro lado son de color oscuro 9. Pez cebra adulto robustamente prefieren el lado oscuro de la arena bajo condiciones de control 6,9,13. El aumento de la ansiedad se define operacionalmente como significativamente más tiempo de permanencia en la zona oscura, y la disminución de la ansiedad se puede suponer cuando el pez pasa relativamente más tiempo de permanencia en la zona de luz. Con el software de seguimiento de movimiento, otras variables informativas también se pueden cuantificar, incluyendo velocidad media, la inmovilidad, meandros, y la zona de transiciones 14. El método de dosificación desarrollado en nuestro laboratorio puede aplicarse a cualquier investigación en el que los compuestos solubles en agua se administran a uno o más zebrafish. Muchos otros agentes farmacológicos que pueden beneficiarse de esta metodología en la actualidad se están probando en el pez cebra. Comúnmente compuestos ensayados incluyen la nicotina, clordiazepóxido, buspirona, y escopolamina, que se disolvió en una manera similar a etanol; mezclando la cantidad apropiada de la sustancia química en el agua. Por lo tanto, el alcance general de este procedimiento es mucho más amplio y no limitado a etanol. Además, después de la dosificación con fármacos para múltiples días, la tarea luz / oscuridad es sólo una de las muchas pruebas de comportamiento que podrían emplearse. Después de la administración de drogas o durante la retirada, otros ensayos populares que se pueden utilizar incluyen la prueba de buceo tanque de novela 15 y las pruebas de comportamiento social, tales como shoaling 16. El siguiente procedimiento se esbozarán un método eficiente de transferir repetidamente grupos de peces o peces individuales en soluciones que contienen un compuesto farmacológico de interés. Además, el proceso de las pruebas de la ansiedad con la prueba de luz / oscuridaden grupos de peces que están en retirada después de ser expuesto a largos horarios de la administración de alcohol se describirá.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Three Shelf benchtop housing system</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/research-systems/z-hab-system/">http://aquatichabitats.com/research-systems/z-hab-system/</a></td>
		</tr>
		<tr>
			<td>1.5L Spawning tank w/400 micron baffle</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/">http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/</a></td>
		</tr>
		<tr>
			<td>Pure Grain Ethanol</td>
			<td>Luxco, INC</td>
			<td>N/A</td>
			<td><a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;aid=3759149&amp;store=Scientific">http://www.luxco.com/public/brands/brands.asp?brandid=21</a></td>
		</tr>
		<tr>
			<td>Ethovision XT Motion tracking software</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt">http://www.noldus.com/animal-behavior-research/products/ethovision-xt</a></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Canada</td>
			<td></td>
			<td><a href="http://www.eppendorf.ca/int/index.php?l=211&amp;pb=e396f9e705e754a3&amp;action=products&amp;contentid=1&amp;catalognode=9477.">http://www.eppendorf.ca/int/index.php?l=211&#38;pb=e396f9e705e754a3&#38;action=products&#38;contentid=1&#38;catalognode=9477.&#160;</a></td>
		</tr>
		<tr>
			<td>Light/Dark Arena</td>
			<td>Custom</td>
			<td></td>
			<td>Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long</td>
		</tr>
	</tbody>
</table>

a novel method of drug administration to multiple zebrafish (danio rerio) and the quantification of withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

Para mantener la precisión y el control en estudios farmacológicos con el pez cebra es importante el tiempo de la duración de la administración de etanol consistentemente y con precisión como se describe anteriormente. Nuestro procedimiento puede aumentar la facilidad y el rendimiento del procedimiento de dosificación. La administración de etanol en la fecha prevista o bien un binge-semanal o diaria de moderada resultó en niveles alterados de ansiedad, medida con el / test oscuro luz, en comparación con los con...

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Un nuevo método de la Administración de Drogas a múltiple pez cebra (<em&gt; Danio rerio</em&gt;) Y la cuantificación de Retiro

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

a-novel-method-drug-administration-to-multiple-zebrafish-danio-rerio

Research

JoVE Journal

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

12.0K vistas.  MacEwan University. A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Video: A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Evaluación de las vocalizaciones de ultrasonidos durante autoadministración de drogas en ratas

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálisis de etanol durante etanol operante determinación y la auto-administración de etanol por cromatografía de gases

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Puñalada de Lesiones de la herida del pez cebra Telencéfalo adultos: un método para investigar Vertebrados Cerebro neurogénesis y Regeneración

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Evaluación de la Viabilidad Neuronal Utilizando Colorante de Fluoresceína-Ioduro de Propidio Doble Coloración en Gránulos Cerebelosos Cultura de Neuronas

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Un nuevo método para modelar la encefalopatía traumática crónica<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Modelo de una rata de la fatiga Central mediante la método de plataforma múltiple

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Un método volumétrico para la cuantificación de vasoespasmo Cerebral en un modelo murino de hemorragia subaracnoidea

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Autorradiografía como un método sencillo y potente para la visualización y caracterización de dianas farmacológicas

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...