SG wird durch Mittel aus der State University of New York at Buffalo und von John R. Oishei Foundation unterstützt.

<html> 1. Vorbereitung der Reagenzien <ol><li> Bereiten Sie die PEMFA Buffer mit 100 mM Pipes, 2 mM EGTA und 1 mM MgSO 4. Den pH-Wert des Puffers auf 7,0. Die PEMFA Puffer sterilfiltriert und bei 4 ° C. </li><li> Bereiten PBT, indem 1 ml Triton X-100 bis 100ml 1x PBS (Phosphate Buffered Saline) (pH 7,0). Lagerung bei 4 ° C. </li><li> Bereiten Sie 5% BSA (Rinderserumalbumin) mit 0,01% NaAcide in 1x PBS (pH 7,0). </li><li> Bereiten Sie die Salzlösung mit 0,7% NaCl und 0,03% Triton X-100 in entionisiertem Wasser. </li><li> Bereiten Sie das Fixativ durch die Kombination von 4 Teilen PEMFA Puffer mit 1 Teil 37% Formaldehyd. </li></ol> 2. Sammlung von Embryos <ol><li> Bereiten Sie einen Käfig für die Drosophila-Stamm von Interesse, indem sie&gt; 100 fliegt in einer Kunststoff-Flasche mit einem Adapter mit Agar Fütterung Platte. </li><li> Lassen Sie den Käfig fliegt im Dunkeln bei Raumtemperatur für 72 Stunden, wechselnden Platten alle 8-12 Stunden, so dass die Fliegen, um den Käfig zu bekommen gewöhnt und halten nicht ihre Eier. </li><li> Für das Experiment sammeln Embryonen für 12 Stunden. Entfernen Sie die Petrischale aus dem Behälter und ersetzen Sie sie. Nehmen Sie die Petrischale entfernt und waschen die Embryonen von der Schüssel mit der Kochsalzlösung und einem sauberen Pinsel. </li><li> Sammeln Sie die Embryonen in ein feines Sieb passieren. Spülen mit Kochsalzlösung, um überschüssige Hefe zu entfernen. </li><li> Mit einem Spatel vorsichtig abholen Embryonen und legen Sie sie in eine kleine Glasflasche mit einer Kappe. Fügen Sie zu 1 ml Kochsalzlösung. </li></ol> 3. Die Entfernung des Chorion und Dottergangzyste Membranen und Fixation <ol><li> Nach der Sammlung von Embryonen, bereiten Sie die Heptan-Fix-Lösung durch die Kombination von 50% Heptan und 50% Fixativ. Die Lösung wird mit Heptan oben und das Fixativ auf dem Boden geschichtet werden. </li><li> In dem Glasfläschchen waschen die Embryonen durch Inkubation in der Salzlösung für 5 Minuten. Entfernen Sie das überschüssige Salz mit einer Pasteurpipette. </li><li> Spülen Sie die Embryonen mit entionisiertem Wasser. </li><li> Dechorionate die Embryonen durch Inkubation in 50% Bleichmittel für 3 Minuten. </li><li> Spülen Sie den Embryonen gründlich mit entionisiertem Wasser. </li><li> Fix die Embryonen durch Inkubation für 30 Minuten in der Heptan-Fix-Lösung unter leichtem Schütteln. </li><li> Mit einer Pasteurpipette ersetzen die Fixiermittel (unterste Schicht) mit Methanol Verlassen des Heptan in die Röhre. Kräftig schütteln, um die Dotterhaut zu entfernen. Die devitellinized Embryonen wird am Ende der Röhre zu versenken. </li><li> Sammeln Sie die Embryonen in eine neue Glasflasche mit einer Pasteurpipette. Spülen Sie den Embryonen fünfmal mit Methanol, inkubieren jedes Mal die Embryonen für 5 Minuten. </li></ol> 4. Antikörper-Färbung, Montage-und Visualisierungs- <ol><li> Rehydrieren die Embryonen in einem Gemisch aus 50% PBT-Lösung und 50% Methanol für 5 Minuten. Dann in 100% PBT-Lösung rehydriert für 5 Minuten. </li><li> Blockieren Sie die Embryonen durch Inkubation in 5% BSA mit 0,01% NaAcide Lösung für 1 Stunde bei 4 ° C. </li><li> Embryonen sind nun bereit für die Antikörper-Färbung. Bereiten Sie den primären Antikörper durch Verdünnen auf die geeignete Konzentration mit 5% BSA mit 0,01% NaAcide Lösung. Wir verwenden ein Antikörper gegen das Cystein-String-Protein (CSP), eine vesikuläre synaptischen Protein und ein Anti-HRP oder Meerrettichperoxidase-Antikörper, der eine neuronale spezifische-Kohlenhydratanteil in der Oberfläche Glykoproteine ​​und Etiketten aller Neurone erkennt. </li><li> Inkubieren Sie die Embryonen über Nacht bei 4 ° C in den primären Antikörper-Lösung. </li><li> Am folgenden Tag, waschen Sie die Embryonen 10 Mal mit PBT über den Verlauf einer Stunde. </li><li> Bereiten Sie den sekundären Antikörper-Lösung durch Verdünnung der entsprechenden Konzentration. Wir verwenden Alexa Sekundärantikörper in einer Konzentration von 1:200. </li><li> Inkubieren Sie die Embryonen für 2 Stunden in 4 ° C in den sekundären Antikörper-Lösung. </li><li> Waschen Sie die Embryonen 10 Mal mit PBT über den Verlauf einer Stunde. </li><li> Inkubieren Sie die Embryonen über Nacht in Vectashield Eindeckmedium. </li><li> Montieren Sie die Embryonen auf einem Glasträger und Dichtung das Deckglas mit Nagellack. Visualisieren Embryonen mit einem Fluoreszenzmikroskop. </li></ol> 5. Repräsentative Ergebnisse <img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="Abbildung 1" /> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="Abbildung 1" /> HRP-FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="Abbildung 1" /> Zusammengeführt Abbildung 1: Eine repräsentative Bilder, die die embryonalen ZNS im Stadium 16-17, mit Antikörpern gegen CSP (rot) und HRP (grün). Beachten Sie die unterschiedliche Färbung der Kommissur. <img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="Abbildung 2" /> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="Abbildung 2" /> HRP-FITC &quot;/&gt; Zusammengeführt Abbildung 2: Eine repräsentative Bilder, die die embryonale PNS bei Stufe 16-17 mit Antikörpern gegen CSP (rot) und HRP (grün). Beachten Sie die sich wiederholenden Mustern von peripheren Neuronen.

<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>

Die Drosophila-Embryo ist ein leistungsstarkes System für die Untersuchung der zellulären und molekularen Veränderungen während der neuronalen Entwicklung. In diesem Protokoll haben wir gezeigt, wie man ganze Berg Embryonen für die Immunhistochemie vorzubereiten. Wir haben dieses Protokoll aus dem Protokoll in Carroll und Scott, 1985, beschrieben angepasst. Dieses Protokoll kann auch angepasst werden andere neuronale Antikörper-Test, sondern die Optimierung von Antikörpern getan werden sollte. Darüber h...

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<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-25</td>
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<td>CSP</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
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<td>HRP-FITC</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch Laboratories</td>
<td>323-095-021</td>
<td>&nbsp;</td>
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<td>Alexa Fluor 568 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004</td>
<td>&nbsp;</td>
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<td>Vectashield Mounting Medium</td>
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visualization of the embryonic nervous system in whole-mount drosophila embryos

Die Drosophila-Embryo ist ein attraktives Modell für die Untersuchung der zellulären und molekularen Grundlagen der neuronalen Entwicklung. Hier beschreiben wir das Verfahren für die Visualisierung von Drosophila embryonalen Nervensystems mit Antikörpern gegen neuronale Proteine. Da die gesamte embryonalen peripheren Nervensystems und des zentralen Nervensystems sind gut auf der Ebene der einzelnen Zellen (..; Bodmer et al, 1987;. Dambly-Chaudière et al, 1986 Bodmer et al, 1989) charakterisiert, jede Aberrationen zu diesen Systemen können leicht identifiziert werden mit Antikörpern gegen verschiedene neuronale Proteine. Die Embryonen sind zu bestimmten Zeiten gesammelt, um sicherzustellen, dass die Embryonen in die richtige Entwicklungsstadien für die Visualisierung sind. Nach dem Sammeln werden die äußeren Schichten des Embryos, das Chorion Membran und der Dotter-Umschlag, der den Embryo umgibt, vor der Fixierung entfernt. Die Embryonen werden dann mit neuronalen Antikörpern und visualisiert mit fluoreszenzmarkierten Sekundärantikörper. Embryos in den Stadien 12-17 visualisiert werden, um die embryonalen Nervensystems zugreifen. Auf Stufe 12 des ZNS Keimstreifs beginnt Verkürzung und von Stufe 15 die endgültige Muster der Kommissur erreicht worden ist. Mit Stufe 17 des ZNS Verträge und die PNS ist voll erschlossen (Campos-Ortega et al. 1985). So Veränderungen in der Struktur des PNS und ZNS können leicht in diesen Entwicklungsstadien beobachtet werden.

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

Wir beschreiben das Verfahren zur Vorbereitung inszeniert<em&gt; Drosophila</em&gt; Embryonen für die Visualisierung des embryonalen Nervensystems während der Embryogenese.

Visualisierung des embryonalen Nervensystems 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 ...

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

Research

JoVE Journal

Neuroscience

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

13.1K Aufrufe.  SUNY-University at Buffalo. Wir beschreiben das Verfahren zur Vorbereitung inszeniert Drosophila Embryonen für die Visualisierung des embryonalen Nervensystems während der Embryogenese.

Serie: 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.

Ganze Berge Vorbereitung des Adult Drosophila Bauchmark für Giant Fiber Injection Dye

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

Forschung

Neurowissenschaften

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.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

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.

Beschriften einzelner Zellen im zentralen Nervensystem<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.

Eine Verletzung Paradigm Central Nervous System Repair in Untersuchen<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.

Direkt Intraventrikuläre Abgabe von Arzneimitteln an die Nager 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 ...

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.

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

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 optogenetische Stimulation des Nager 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 ...

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.

Ganze Berge Kennzeichnung von Cilia im Hauptriechsystem der Mäuse

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 des enterischen Nervensystems

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.

Eine einfache Ein-Schritt-Dissection-Protokoll für Whole-mount Vorbereitung von Adult<em&gt; Drosophila</em&gt; Brains

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