El trabajo fue apoyado por un premio de Desarrollo Científico Clínico de los NIH K08NS002083, un Instituto de Investigación Shervert Frazier Grant, y un premio Young Investigator NARSAD a Edgar A. Büttner.

1. Retraso en el desarrollo / letalidad Ensayo: Las dos cepas mutantes (Mut1 y Mut2) de tipo salvaje (N2) y se ponen a prueba en tres Clozapina Las concentraciones en una placa de 12 pocillos <ol><li> El día 1, vierta 2 ml NGM medio 10 en cada pocillo de una placa de 12 pocillos (cada pozo con un diámetro de 2 cm) y se deja endurecer en el banquillo a temperatura ambiente (TA) durante la noche. El mismo día, elegir una colonia de bacterias Escherichia coli OP50, infectar a una botella de 50 ml de solución de LB, y la cultura en un agitador de 37 ° C a 220 rpm de rotación durante la noche. </li><li> En el día 2, transferir 20 l de cultivo bacteriano OP50 en el centro de cada NGM bien. Incubar las placas en una incubadora a 37 ° C o dejarlos a temperatura ambiente durante la noche para permitir que el césped bacteriano a crecer más grueso. </li><li> Haga una solución de clozapina de stock 80 mM disolviendo la clozapina en DMSO (dimetilsulfóxido) solvente. A continuación, hacer una solución de trabajo de 80 l con la solución madre diluida en 1,7 mM de ácido acético basándose en la Tabla 1. Nota: La solución de trabajo para un fármaco particular de interés debe ser determinada por hacer una serie de pruebas preliminares de concentración para encontrar la concentración más apropiado para distinguir el tipo salvaje de los mutantes. </li><li> Transferencia de 80 l clozapina solución de trabajo en la placa de NGM sembrado bien y agitar la placa para permitir que la solución para distribuir de manera uniforme sobre la superficie. Espere a que la placa se seque. Utilice la placa para el ensayo en el mismo día o colocarlo en una incubadora de 20 ° C para su uso al día siguiente. Nota: La solución de trabajo es una suspensión del fármaco debido a la baja solubilidad de la clozapina, especialmente a mayor concentración. Por lo tanto, vórtice el tubo antes de transferir la solución a la placa de fármaco con el fin de garantizar que la concentración de fármaco es exacta. </li><li> Lavar los gusanos de un plato de 3,5 cm que contiene muchos adultos grávidas con tampón M9 y luego girar hacia abajo en un tubo de 15 mla 2.000 rpm durante 1 min. </li><li> Aspirar el sobrenadante. Añadir 5 ml de solución de lejía (NaOH: hipoclorito: ddH2O a 4:01:05) y desbaratar los nematodos invirtiendo suavemente los tubos. </li><li> Observar a los animales hasta la mitad de ellos se disuelven en torno al 5 min. Centrifugar los huevos a 2.000 rpm durante 1 min. </li><li> Aspirar el sobrenadante y añadir 14 ml de buffer M9 rápidamente. </li><li> Centrifugar los huevos a 2000 rpm durante 1 min y aspirar el sobrenadante, dejando aproximadamente 100 l solución. Vortex para suspender los huevos. </li><li> Transferencia de 2 l huevos suspendidos en M9 en un plato regular de NGM para probar cuántos huevos se transfieren. Ajustar el volumen de los huevos en suspensión para asegurar que hay aproximadamente 30-35 huevos en cada transferencia. Transferencia 30-35 huevos a cada pocillo de la placa de ensayo, y luego incubar en una incubadora de 20 º C durante 24 h. </li><li> En el primer día del ensayo, anotar el número de huevos eclosionados. Ajuste el número total de animales que nacieron para 25/well por Pickión de los gusanos adicionales si es necesario. </li><li> En los días siguientes, observar a los animales y la puntuación de ellos cada 24 horas. El grado de desarrollo se obtuvo sobre la base del tamaño del animal y la forma de la vulva 11. El experimento dura 5-6 días con el efecto más robusto típicamente visto en la tercera o cuarta día. </li><li> Ingrese los datos en un archivo de Excel y calcular el porcentaje de animales en cada etapa de desarrollo para todos los días del experimento. Generar gráficos con la función de &#39;100% Stacked columna &#39;en &#39;2 D-columna &quot;del menú. </li></ol> 2. Faríngea Ensayo de Bombeo: animales adultos jóvenes y para los de tipo salvaje y cepas mutantes se califican en placas Clozapina ensayo <ol><li> La placa de ensayo se obtiene siguiendo el mismo protocolo utilizado para el ensayo de retardo / letalidad del desarrollo. </li><li> Elige 50 animales L4 para cada cepa a NGM placas sembradas 24 horas antes del ensayo y se incuba a los gusanos a 20 ° C. </li><li> Elige 10 animales a un ASSAy placa también. A continuación, elija 10 animales al siguiente bien cada 15-20 min. </li><li> Después de que los animales en el primer pozo han sido expuestos a drogas durante 30 min, iniciar el ensayo mediante la observación de bombeo faríngea bajo un microscopio de disección y la puntuación de las bombas durante 20 s 9. Una vez que el bombeo de la faringe se anotó, recoger el gusano de la placa. </li></ol>

<ol>
	<li>Donohoe, D. R., Jarvis, R. A., Weeks, K., Aamodt, E. J., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+adaptation+in+C.+elegans+produced+by+antipsychotic+drugs+requires+serotonin+and+is+associated+with+calcium+signaling+and+calcineurin+inhibition.">Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.</a> Neurosci. Res. 64, 280-289 (2009).</li><li>Karmacharya, 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=Clozapine+interaction+with+phosphatidyl+inositol+3-kinase+(PI3K)/insulin-signaling+pathway+in+Caenorhabditis+elegans.">Clozapine interaction with phosphatidyl inositol 3-kinase (PI3K)/insulin-signaling pathway in Caenorhabditis elegans.</a> Neuropsychopharmacology. 34, 1968-1978 (2009).</li><li>Rand, J. B., Johnson, C. D., Estein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Carnorhabditis elegans Modern Biological Analysis of an Organism. 48, 187-204 (1995).</li><li>Kwok, T. 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=A+small-molecule+screen+in+C.+elegans+yields+a+new+calcium+channel+antagonist.">A small-molecule screen in C. elegans yields a new calcium channel antagonist.</a> Nature. 441, 91-95 (2006).</li><li>Wang, X., Sliwoski, G. R., Buttner, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+Caenorhabditis+elegans+genetics+for+understanding+human+psychiatric+disease.">The relevance of Caenorhabditis elegans genetics for understanding human psychiatric disease.</a> Harv. Rev. Psychiatry. 19, 210-218 (2011).</li><li>Donohoe, D. R., Aamodt, E. J., Osborn, E., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antipsychotic+drugs+disrupt+normal+development+in+Caenorhabditis+elegans+via+additional+mechanisms+besides+dopamine+and+serotonin+receptors.">Antipsychotic drugs disrupt normal development in Caenorhabditis elegans via additional mechanisms besides dopamine and serotonin receptors.</a> Pharmacol. Res. 54, 361-372 (2006).</li><li>Karmacharya, 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=Behavioral+effects+of+clozapine:+involvement+of+trace+amine+pathways+in+C.+elegans+and+M.+musculus.">Behavioral effects of clozapine: involvement of trace amine pathways in C. elegans and M. musculus.</a> Brain Res. 1393, 91-99 (2011).</li><li>Saur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans+Identifies+the+Nicotinic+Acetylcholine+Receptor+Subunit+ACR-7+as+an+Antipsychotic+Drug+Target.">A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target.</a> PLoS Genet. 9, (2013).</li><li>Avery, L., You, Y. 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Los autores declaran que no existe conflicto de interés involucrados.

A continuación, describimos métodos para probar los efectos de la APD sobre el desarrollo y el comportamiento de C. elegans. DMSO o etanol se usa para disolver la clozapina, ya que el fármaco es relativamente insoluble en agua. Debido a que los disolventes se han reportado para afectar C. elegans biología 12, DMSO-solas o grupos de control de etanol por sí sola, son esenciales. La concentración más alta de DMSO utilizada en nuestros ensayos es hasta 3%, que no tiene un efecto obvio so...

Caenorhabditis elegans es un organismo genético simple susceptibles de pantallas genéticos directo e inverso a gran escala y pantallas genéticos químicos. C. elegans es sensible a un amplio espectro de compuestos bioactivos y por lo tanto se ha utilizado con éxito para definir los mecanismos de acción de una variedad de tales compuestos. Por ejemplo, compuestos bioactivos estudiados usando la farmacogenética gusano incluyen agonistas del receptor de acetilcolina (por ejemplo, levamisol, nicotina, morantel, y pirantel), anestésicos (por ejemplo, halotano), cafeína, inhibidores de la colinesterasa (por ejemplo, el aldicarb, LANNATE, y triclorfón), fluoruro, el GABA-relacionados compuestos (por ejemplo, GABA y muscimol), ivermectina, paraquat, ésteres de forbol y fármacos relacionados con la serotonina (por ejemplo, la serotonina y imiprimine) 3. Además, C. elegans se ha utilizado para pantallas de molécula pequeña a gran escala, lo que permite el descubrimiento de nueva compuesto bioactivos y la identificación de dianas genéticas nuevas 4. El C. elegans genoma incluye fármaco potencial antipsicótico (APD) objetivos conservadas en los seres humanos, incluidos los genes que codifican las proteínas necesarias para la síntesis de neurotransmisores y de la estructura y función sináptica 5. Por lo tanto, C elegans neurogenetics y métodos ofrecen la neurobiología para descubrir nuevos mecanismos moleculares de la acción de la APD. En los nematodos, la exposición APD en el desarrollo temprano produce retraso en el desarrollo, y en concentraciones más altas, la letalidad de 2,6. Exposición APD durante la edad adulta produce fenotipos conductuales. Por ejemplo, la exposición a la clozapina inhibe la locomoción y el bombeo faríngeo y mejora la postura de huevos 1,2,7. Retraso en el desarrollo inducido por APD y letalidad son los fenotipos de gran alcance para las pantallas de genética química a gran escala. Estos fenotipos son complejos en la medida en que es probable que tengan más de un basi celulares y genéticoss. Por lo tanto, se espera que tales pantallas genéticas para producir una variedad de dianas de medicamentos indirectos. Sin embargo, nuestro laboratorio ha realizado candidatos pantallas de genes y una pantalla de RNAi de todo el genoma de los supresores de retraso en el desarrollo inducido por APD y la letalidad y ha genes que probablemente codifican blancos directos, incluyendo la dopamina, la insulina y los receptores nicotínicos de la acetilcolina 2,8 recuperado con éxito. Pantallas genéticas basadas en los comportamientos inducidos por el APD en el adulto también han conducido a la identificación de los objetivos de APD novedosas, y ahora estamos validando dianas de las pantallas tanto de desarrollo y de comportamiento en los mamíferos 7. Por lo tanto, un enfoque genético químico de invertebrados para descubrir nuevos mecanismos moleculares de acción de APD parece ser factible 5,8. El C. elegans faringe es un órgano que incluye 20 neuronas, las células musculares 20, y 20 células accesorias, envueltos por una membrana basal. Similar al corazón de los mamíferos, la faringe es un autónomaND constantemente Bombas de alimentación desde el medio ambiente externo 9. La inhibición de la velocidad de bombeo de la faringe compromete la absorción de los alimentos, y por lo tanto las mutaciones o medicamentos que inhiben la faringe de bombeo causa retraso en el desarrollo o la detención 9. APD inhiben la velocidad de bombeo de la faringe, lo que representa, en parte, por sus efectos sobre el desarrollo y viabilidad 1,2. Aquí, nosotros usamos la clozapina APD atípica como ejemplo para demostrar los ensayos de medicamentos para el desarrollo del nematodo y el bombeo de la faringe.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

Caenorhabditis elegans es un organismo genético simple susceptible a gran escala de avance y retroceso genéticos pantallas y pantallas genéticos químicos. El C. elegans genoma incluye fármaco potencial antipsicótico (APD) objetivos conservadas en los seres humanos, incluidos los genes que codifican las proteínas necesarias para la síntesis de neurotransmisores y de la estructura y la función sináptica. Exposición APD produce una demora y / o mortalidad en los nematodos en el desarrollo de una manera dependiente de la concentración. Estos fenotipos son causados, en parte, por la inhibición inducida por APD de faríngea de bombeo 1,2. Por lo tanto, el fenotipo de desarrollo tiene una base neuromuscular, lo que es útil para estudios de farmacogenética de los neurolépticos. Aquí demostramos procedimientos detallados para probar los efectos de APD sobre el desarrollo del nematodo y el bombeo de la faringe. Para el ensayo de desarrollo, los embriones sincronizados se colocan en medio de crecimiento de nematodos (NGM) placas que contienen APD, y las etapas de desarrollo de Animales son entonces marcados diaria. Para el ensayo de velocidad de bombeo de la faringe, protagonizaron los animales adultos jóvenes se ponen a prueba en placas NGM contienen APD. El número de bombas faríngeas por unidad de tiempo se registra, y se calcula la tasa de bombeo. Estos ensayos se pueden usar para el estudio de muchos otros tipos de moléculas pequeñas o incluso moléculas grandes.

1. El retraso del desarrollo / resultado del ensayo de letalidad Un resultado típico para el ensayo de retardo / letalidad del desarrollo se demuestra en las Figuras 1a y 1b. Cuando los animales de tipo salvaje en el grupo de control se han convertido en la etapa adulta grávida (Figura 1a), de tipo salvaje animales expuestos a la clozapina se encuentran todavía en las etapas jóvenes larvas o están muertos (Figura 1b). Figura 1c</st...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Enfoques para probar los efectos de los fármacos antipsicóticos (APD) en Caenorhabditis elegans se demuestran. Los ensayos se describen para las pruebas de efectos del fármaco sobre el desarrollo y la viabilidad y sobre la tasa de bombeo de la faringe. Estos métodos se aplican también para los experimentos farmacogenéticos con clases de fármacos diferentes a APD.

Métodos para el estudio de los mecanismos de acción de los fármacos antipsicóticos en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

methods-for-studying-mechanisms-action-antipsychotic-drugs

Research

JoVE Journal

Neuroscience

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

11.4K vistas.  Harvard Medical School.  Enfoques para probar los efectos de los fármacos antipsicóticos (APD) en Caenorhabditis elegans se demuestran. Los ensayos se describen para las pruebas de efectos del fármaco sobre el desarrollo y la viabilidad y sobre la tasa de bombeo de la faringe. Estos métodos se aplican también para los experimentos farmacogenéticos con clases de fármacos diferentes a APD. 

Video: Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Del ratón en el útero de electroporación: Transefection controlada Gene espacio-temporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Investigación

Neurociencias

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular directa de medicamentos al roedor Sistema Nervioso Central

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

La<em&gt; Caenorhabditis elegans</em&gt; Sistema Modelo para el Estudio Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un ensayo para medir los efectos del etanol sobre la velocidad de locomoción<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microcámaras de agarosa para Imagen de calcio a largo plazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Las métricas para caracterizar nuevos embrionarias Alargamiento del nematodo<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UN<em&gt; Caenorhabditis elegans</em&gt; Ensayo de aversión al cobre basado en el estado nutricional

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagen radiométrica calcio de neuronas individuales en comportamiento Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Imágenes de espinas dendríticas en Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...