Este estudio fue apoyado por el programa apoyado por el MEXT para la Fundación para la Investigación Estratégica en las Universidades Privadas y JSP Young Scientist B subvención para HT Nos gustaría dar las gracias a Ohtake Norihito para editar los clips de vídeo.

1. Preparar plato Manzana <ol><li> Mezclar 12,5 g de agar, 125 ml de jugo 100% disponible en el mercado de la manzana, 12,5 g de glucosa, y 375 ml de H 2 O. Microondas la mezcla durante 1 a 2 minutos y se vierte a la placa de cultivo de 3 cm de células. Almacenar a 4 ° C. </li></ol> 2. Cruzamiento genético <ol><li> Utiliza el siguiente cruce genético representativo: </li></ol> Or49a-mCD8 :: GFP / CyO x w 1118 3. Protocolo de disección y la tinción <ol><li> Se anestesia la marcha y luego se corta la cabeza mosca vertical sujetándolo mediante un fórceps. </li><li> Coloque con cuidado la parte que contiene las antenas en una placa de manzana. </li><li> Cortar el tercer segmento de la antena con tijeras de disección finas. </li><li> Coloque 90 l de la solución de fijación (4% de paraformaldehído en 0,1% de PBST (PBS con 0,1% de Triton X-100)) a la mitad de una placa de cultivo con fondo de vidrio. </li><li> Transferir suavemente la antenn diseccionadoae con una aguja directamente a la solución de fijación. Si es necesario, sumergir físicamente las antenas en la solución usando la aguja. </li><li> Incubar durante 40 minutos a temperatura ambiente (RT). Lavar las antenas en 0,4% de PBST (PBS con 0,4% de Triton X-100), 3x 10 minutos en cada uno, mantenerlos en el mismo plato. Utilice puntas amarillas de quitar y añadir solución PBST. Utilice 90 l de la solución de lavado en cada ocasión. NOTA: No coloque el plato en una coctelera durante inmunohistoquímica. Antes de la eliminación o la adición de la solución en el plato, llevar a todos las antenas en el centro de la cápsula, utilizando la aguja y agregar o quitar cuidadosamente la solución desde el borde del plato. </li><li> Bloquear las antenas con 90 l de 5% de suero de caballo normal en 0,1% de PBST durante 20 minutos a TA. </li><li> Después de retirar la solución de bloqueo, incubar las antenas con 90μl de anticuerpos primarios en 0,1% PBST que contenía 5% de suero de caballo durante 48 horas a 4 ° C en un recipiente húmedo como se describe anteriormente <sarriba&gt; 8. </li><li> Lávese las antenas 6x 10 minutos en el 0,4% PBST. </li><li> Incubar las antenas con 90 l de anticuerpos secundarios en 0,1% PBST que contiene 5% de suero de caballo durante 48 horas a 4 ° C. Lave 6x 10 minutos usando 0,4% PBST. </li><li> Para montar la antena, eliminar PBST de la placa de cultivo tanto como sea posible e introducir gradualmente dos concentraciones diferentes de glicerol a las antenas. En primer lugar añadir 40% de glicerol a la placa durante 1 a 2 minutos; luego quite esto y agregar 80% de glicerol. </li><li> Extraer con cuidado la antena (incluyendo el 80% de glicerol) de la placa de cultivo utilizando la punta amarilla y colocarlos en un portaobjetos. Con suavidad, coloque un cubreobjetos encima y sellar los bordes cubreobjetos con esmalte de uñas. Las antenas están ahora listos para ser fotografiado por microscopía de fluorescencia. </li></ol>

<ol>
	<li>Christensen, T. A., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Representation of olfactory information in the brain In The Neurobiology of Taste and Smell. , 201-232 (2000).</li><li>Ache, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+common+strategy+for+transducing+olfactory+information.">Towards a common strategy for transducing olfactory information.</a> Sem. Cell Biol. 5, 55-63 (1994).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction.">Odorant-selective genes and neurons mediate olfaction.</a> C. elegans. Cell. 13, 515-527 (1993).</li><li>Barth, A. L., Justice, N. J., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchronous+onset+of+odorant+receptor+expression+in+the+developing+zebrafish+olfactory+system.">Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system.</a> Neuron. 16, 23-34 (1996).</li><li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li><li>Stockinger, P., 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=Neural+circuitry+that+governs+Drosophila+male+courtship+behavior.">Neural circuitry that governs Drosophila male courtship behavior.</a> Cell. 121, 795-807 (2005).</li><li>Endo, K., 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=Chromatin+modification+of+Notch+targets+in+olfactory+receptor+neuron+diversification.">Chromatin modification of Notch targets in olfactory receptor neuron diversification.</a> Nat Neurosci. 15, 224-233 (2011).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. , (2011).</li><li>Suh, G. 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=A+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila.">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li><li>Sachse, S., Galizia, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+inhibition+for+temporal+and+spatial+odor+representation+in+olfactory+output+neurons:+A+calcium+imaging+study.">Role of inhibition for temporal and spatial odor representation in olfactory output neurons: A calcium imaging study.</a> J Neurophysiol. 87, 1106-1117 (2002).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117, 965-979 (2004).</li><li>Vosshall, L. B., Wong, A. M., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+in+the+fly+brain.">An olfactory sensory map in the fly brain.</a> Cell. 102, 147-159 (2000).</li><li>Couto, A., Alenius, M., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular,+anatomical+and+functional+organization+of+the+Drosophila+olfactory+system.">Molecular, anatomical and functional organization of the Drosophila olfactory system.</a> Curr Biol. 15, 1535-1547 (2005).</li><li>Clyne, P., 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=Odorant+response+of+individual+sensilla+on+the+Drosophila+antenna.">Odorant response of individual sensilla on the Drosophila antenna.</a> Invert Neurosci. 3, 127-135 (1997).</li><li>Vosshall, L. B., 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+spatial+map+of+olfactory+receptor+expression+in+the+Drosophila+antenna.">A spatial map of olfactory receptor expression in the Drosophila antenna.</a> Cell. 96, 725-736 (1999).</li><li>Wang, J. W., 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=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112, 271-282 (2003).</li></ol>

Los autores no tienen nada que revelar.

La disección de la antena de Drosophila se describe es simple y fácil de realizar en un entorno de laboratorio. Para garantizar una disección éxito, es esencial utilizar las tijeras bien afiladas. Mientras que la inmunotinción la antena diseccionado, es importante incubar en un recipiente lleno de humedad para evitar la evaporación de la solución de anticuerpo. La antena diseccionado tiene una tendencia a flotar en la solución. Uso de 0,1% de Triton en PBS durante la fijación y el bloqueo de pasos faci...

El sistema olfativo se utiliza para distinguir entre una inmensa variedad de moléculas de olor y posteriormente enviar la información resultante a los centros superiores del cerebro. Esta entrada se utiliza para controlar con precisión los comportamientos animales fundamentales, como la alimentación y el apareamiento 1-6. Como cada tipo de neurona olfativa está asociado a un conjunto específico de los olores, la diversificación de las neuronas receptoras olfativas (ORN) s es esencial para la función del sistema olfativo adecuada 7. Genética de Drosophila, nos permite llevar a cabo la investigación solo nivel celular que implica mecanismos moleculares asociados al desarrollo de ORN y la función fisiológica 8-16. Whole inmunotinción monte de Drosophila antenas nos ha permitido comprender con mayor detalle los mecanismos moleculares implicados en la diversificación de las neuronas olfativas del receptor (ORN) s 7. Aquí le ofrecemos una amplia descripción de un método sencillo para achieve esto.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Stemi DV4 dissection microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:113px;">Stemi DV4</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Glass bottom culture dishes&#160;</td>
			<td style="width:143px;">MatTek corporation</td>
			<td style="width:113px;">P35G-0-10-C</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Dissection scissor</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:113px;">15000-08</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Rat anti-ELAV</td>
			<td style="width:143px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:113px;">7E8A10</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mouse anti-GFP</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:113px;">A11122</td>
			<td style="width:116px;">Dilution 1:400</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rabbit IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">711-225-152</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rat IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">712-165-150</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
	</tbody>
</table>

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

Las moléculas odoríferas se unen a sus receptores diana de una manera precisa y coordinada. Cada receptor reconoce una señal específica y transmite esta información al cerebro. Como tal, la determinación de cómo se transfiere la información olfativa en el cerebro, tanto la modificación de la percepción y comportamiento, investigación méritos. Curiosamente, hay más pruebas de que los factores de transducción celular y la transcripción están implicados en la diversificación de la neurona olfativa del receptor. Aquí les ofrecemos un conjunto robusto de montaje método de marcaje inmunológico para ensayar in vivo organización neurona olfativa del receptor. Usando este método, se identificaron todas las neuronas olfativas del receptor con el anticuerpo anti-ELAV, un marcador pan-neuronal conocido y Or49a-mCD8 :: GFP, una neurona olfativa del receptor expresa específicamente en Nba neurona utilizando anticuerpo anti-GFP.

Garantizar que tanto la disección y fijación se realizan con rapidez es un factor clave para lograr el éxito con este protocolo. El uso de finas tijeras y pinzas también es crucial. Después de la inmunotinción, las antenas fluorescentes marcadas se examinaron bajo un microscopio confocal. Normalmente nos tomamos secciones 1μm usando una lente de 20x. Hemos marcado Nba 7 ORNs utilizando Or49a-mCD8 :: GFP y contamos el número de Nba ORNs de tipo salvaje antena. El reportero mCD8-GFP se localiza membrana...

Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

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 ...

whole-mount-immunolabeling-olfactory-receptor-neurons-drosophila

Research

JoVE Journal

Neuroscience

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

8.9K vistas.  Doshisha University. 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.

Video: Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualización del sistema nervioso embrionario en Todo el montaje Drosophila Los embriones

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Investigación

Neurociencias

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Preparación Todo el montaje del Adulto Drosophila Cordón nervioso ventral para inyección gigante tinte de fibra

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash fotólisis de compuestos enjaulados en los cilios de las neuronas sensoriales olfativas

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Inducidas Odorante-respuestas registradas de neuronas olfativas del receptor utilizando la técnica de aspiración con una pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Apetitivo aprendizaje asociativo olfativa en<em&gt; Drosophila</em&gt; Las larvas

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

La evaluación de fenotipos neurodegenerativas en<em&gt; Drosophila</em&gt; Las neuronas dopaminérgicas mediante ensayos de Escalada y inmunotinción de todo el cerebro

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Todo el montaje La disección y de inmunofluorescencia del Adulto ratón cóclea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.