SG es apoyado por fondos de la Universidad Estatal de Nueva York en Buffalo y de John R. Oishei Fundación.

<html> 1. Preparación de los reactivos <ol><li> Prepare el tampón PEMFA utilizando canalizaciones con 100 mM, EGTA 2 mM y 1 mM MgSO 4. Ajustar el pH de la solución a 7,0. El buffer PEMFA es esterilizado por filtración y se almacenan a 4 ° C. </li><li> Prepare PBT mediante la adición de 1 ml de Triton X-100 a 100 ml 1x PBS (tampón fosfato salino) (pH 7,0). Almacenar a 4 ° C. </li><li> Preparar 5% de BSA (albúmina de suero bovino) con un 0,01% NaAcide en 1X PBS (pH 7,0). </li><li> Prepare la solución salina con 0,7% NaCl y 0,03% Triton X-100 en agua destilada. </li><li> Preparar el fijador mediante la combinación de 4 partes de búfer PEMFA con formaldehído parte un 37%. </li></ol> 2. Recolección de embriones <ol><li> Prepare una caja para la cepa de Drosophila de interés mediante la colocación de&gt; 100 moscas en una botella de plástico que contiene un adaptador de alimentación con placa de agar. </li><li> Salir de la jaula de las moscas en la oscuridad a temperatura ambiente durante 72 horas, cambio de placas cada 8-12 horas, de modo que las moscas se acostumbren a la jaula y no tienen sus huevos. </li><li> Para el experimento se recogen los embriones durante 12 horas. Retire la placa de Petri del contenedor y reemplazarlo. Tome la placa de Petri quitar y lavar los embriones de la cápsula, utilizando la solución salina y una brocha limpia. </li><li> Recoger los embriones en un colador fino. Enjuagar con solución salina para eliminar cualquier exceso de levadura. </li><li> Usando una espátula suavemente recoger los embriones y colocarlos en un frasco de vidrio con una tapa. Añadir alrededor de 1 ml de solución salina. </li></ol> 3. La eliminación de las membranas Chorion y vitelina y fijación <ol><li> Después de la recolección de embriones, preparar la solución de heptano-Fix mediante la combinación de 50% de heptano y 50% de fijador. La solución será en capas con heptano en la parte superior y el fijador en la parte inferior. </li><li> En el frasco de vidrio lavado de los embriones, mediante su incubación en la solución salina durante 5 minutos. Retire el exceso de solución salina con una pipeta Pasteur. </li><li> Enjuague los embriones con agua destilada. </li><li> Dechorionate los embriones, mediante su incubación en el 50% de lejía durante 3 minutos. </li><li> Enjuague los embriones a fondo con agua destilada. </li><li> Fijar los embriones, mediante su incubación durante 30 minutos en la solución de heptano-Fix con agitación suave. </li><li> Con una pipeta Pasteur reemplazar el fijador (capa inferior) con metanol dejando el heptano en el tubo. Agitar enérgicamente para eliminar la membrana vitelina. Los embriones devitellinized se hunden hasta el fondo del tubo. </li><li> Recoger los embriones en un frasco de vidrio nuevo con una pipeta Pasteur. Enjuague los embriones cinco veces con metanol, cada vez que los embriones se incuban durante 5 minutos. </li></ol> 4. La tinción de anticuerpos, de montaje y visualización <ol><li> Rehidratar a los embriones en una mezcla de 50% de la solución PBT y 50% de metanol durante 5 minutos. Luego rehidratar en un 100% la solución PBT durante 5 minutos. </li><li> Bloquear los embriones, mediante su incubación en 5% de BSA con una solución de NaAcide 0,01% durante 1 hora a 4 ° C. </li><li> Los embriones están listos para la tinción de anticuerpos. Prepare el anticuerpo primario mediante la dilución a la concentración adecuada con 5% de BSA con una solución de NaAcide 0,01%. Nosotros usamos un anticuerpo contra la proteína de cadena de cisteína (CSP), una proteína sináptica vesicular y un anticuerpo anti-HRP rábano o caballo peroxidasa, que reconoce neuronal específica en hidratos de carbono fracción presente en las glicoproteínas de superficie y las etiquetas de todas las neuronas. </li><li> Incubar los embriones durante la noche a 4 ° C en la solución de anticuerpo primario. </li><li> Al día siguiente, lave los embriones 10 veces con PBT en el transcurso de una hora. </li><li> Prepare la solución secundaria de anticuerpos mediante la dilución de la concentración adecuada. Utilizamos anticuerpos Alexa secundaria a una concentración de 1:200. </li><li> Incubar los embriones durante dos horas en 4 ° C en la solución de anticuerpo secundario. </li><li> Lavado de los embriones 10 veces con PBT en el transcurso de una hora. </li><li> Incubar los embriones durante la noche en medio Vectashield montaje. </li><li> Monte de los embriones en un portaobjetos de vidrio y sellar la hoja de la cubierta con esmalte de uñas. Visualizar embriones utilizando un microscopio de fluorescencia. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="Figura 1" /> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="Figura 1" /> HRP-FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="Figura 1" /> Se fusionó Figura 1: Una imagen representativa que muestra el sistema nervioso central embrionarias en la etapa 16-17, el uso de anticuerpos contra la CSP (rojo) y HRP (verde). Tenga en cuenta la coloración distinta de la comisura. <img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="Figura 2" /> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="Figura 2" /> HRP-FITC &quot;/&gt; Se fusionó Figura 2: Un representante de las imágenes que muestra el PNS embrionarias en la etapa 16-17 usando anticuerpos contra la CSP (rojo) y HRP (verde). Tenga en cuenta los patrones repetidos de las neuronas periféricas.

<ol>
	<li>Bodmer, R., Barbel, S., Sheperd, S., Jack, J. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+sensory+organs+by+mutations+of+the+cut+locus+of+D.+melanogaster.">Transformation of sensory organs by mutations of the cut locus of D. melanogaster.</a> Cell. 51, 293-307 (1987).</li><li>Bodmer, R., Carretto, R., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+of+the+peripheral+nervous+system+in+Drosophila+embryos:+DNA+replication+patterns+and+cell+lineages.">Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.</a> Neuron. 3, 21-32 (1989).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Embryonic Development of Drosophila melanogaster. , (1985).</li><li>Carroll, S. B., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+fushi+tarazu+protein+during+Drosophila+embryogenesis.">Localization of the fushi tarazu protein during Drosophila embryogenesis.</a> Cell. 43, 47-57 (1985).</li><li>Dambly-Chaudi&#232;re, C., Ghysen, A., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+connections+between+imaginal+discs+in+Drosophila.">Muscle connections between imaginal discs in Drosophila.</a> Developmental Biology. 113, 288-294 (1986).</li></ol>

El embrión de Drosophila es un potente sistema para la investigación de los cambios celulares y moleculares durante el desarrollo neuronal. En este protocolo se ha demostrado la forma de preparar todo el montaje embriones para inmunohistoquímica. Nos hemos adaptado a partir de este protocolo el protocolo descrito en Carroll y Scott, 1985. Este protocolo también se puede adaptar a otra prueba de anticuerpos neuronales, pero la optimización de los anticuerpos se debe hacer. Además, los defectos del desarrol...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-25</td>
<td>&nbsp;</td>
<tr>
<td>CSP</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HRP-FITC</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch Laboratories</td>
<td>323-095-021</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 568 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium</td>
<td>&nbsp;</td>
<td>Vector Laboratories, Inc.</td>
<td>H-1000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

visualization of the embryonic nervous system in whole-mount drosophila embryos

El embrión de Drosophila es un sistema modelo atractivo para la investigación de las bases celulares y moleculares del desarrollo neuronal. A continuación se describe el procedimiento para la visualización de Drosophila sistema nervioso embrionario utilizando anticuerpos contra las proteínas neuronales. Ya que toda la embrionaria sistemas nervioso periférico nervioso central y están bien caracterizados a nivel de células individuales (Dambly-Chaudière et al, 1986;.. Bodmer et al, 1987;. Bodmer et al, 1989), cualquier aberración que estos sistemas pueden identificar fácilmente el uso de anticuerpos a diferentes proteínas neuronales. Los embriones en desarrollo se reúnen en ciertos momentos para asegurarse de que los embriones se encuentran en las etapas de desarrollo adecuado para la visualización. Después de la recolección, las capas externas del embrión, el corion y la membrana de la envoltura vitelina que rodea el embrión, se retiran antes de la fijación. Los embriones se incuban con anticuerpos neuronales y se visualizan con fluorescencia utilizando anticuerpos secundarios marcados. Los embriones en etapas 12-17 se visualizan para acceder al sistema nervioso embrionario. En la 12 ª etapa de la banda germen del SNC se inicia el acortamiento y la etapa 15 el patrón definitivo de la comisura se ha logrado. Por la etapa 17 de los contratos del SNC y SNP está completamente desarrollado (Campos-Ortega et al. 1985). Así, los cambios en el patrón del SNP y SNC pueden ser fácilmente observados durante estas etapas de desarrollo.

Watch this Scientific Journal Video about Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos at JoVE.com

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

Se describe el procedimiento para preparar etapas<em&gt; Drosophila</em&gt; Embriones para la visualización del sistema nervioso embrionario durante la embriogénesis.

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 ...

visualization-embryonic-nervous-system-whole-mount-drosophila

Research

JoVE Journal

Neuroscience

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

13.2K vistas.  SUNY-University at Buffalo. Se describe el procedimiento para preparar etapas Drosophila Embriones para la visualización del sistema nervioso embrionario durante la embriogénesis.

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

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 ...

Investigación

Neurociencias

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualización de los propioceptores<em&gt; Drosophila</em&gt; Las larvas y pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

El marcaje de células individuales en el Sistema Nervioso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Un paradigma de Lesiones de investigar reparación del sistema nervioso central en<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

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 ...

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 ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenético la estimulación del sistema nervioso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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 ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging del sistema nervioso entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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

Resumen

Explorar más videos

Capítulos en este video

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

Visualización de los propioceptores

El marcaje de células individuales en el Sistema Nervioso Central de

Un paradigma de Lesiones de investigar reparación del sistema nervioso central en

Entrega intraventricular directa de medicamentos al roedor Sistema Nervioso Central

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el

In vivo optogenético la estimulación del sistema nervioso central de roedores

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

In Situ Ca ²⁺ Imaging del sistema nervioso entérico

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto