This study was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities and JSPS Young Scientist B grant for H.T. We would like to thank Ohtake Norihito to edit the video clips.

1. Prepare apple plate
<ol>
	<li>Mix 12.5 g agar, 125 ml 100% commercially available apple juice, 12.5 g glucose, and 375 ml H2O. Microwave the mixture for 1 to 2 minutes and pour to the 3 cm cell culture dish. Store at 4 &#176;C.</li>
</ol>
2. Genetic cross
<ol>
	<li>Use the following representative genetic cross:</li>
</ol>
Or49a-mCD8::GFP/CyO x w1118
3. Dissection and staining protocol
<ol>
	<li>Anesthetize the fly and then cut the fly head vertically by holding it using a forceps.</li>
	<li>Carefully place the antennae containing portion on an apple plate.</li>
	<li>Cut the third segment of the antenna using fine dissection scissors.</li>
	<li>Place 90 &#181;l of the fixation solution (4% paraformaldehyde in 0.1% PBST (PBS with 0.1% Triton X-100)) to the middle of a glass bottom culture dish.</li>
	<li>Gently transfer the dissected antennae with a sharp needle directly to the fixation solution. If necessary, physically submerge the antennae into the solution using needle.</li>
	<li>Incubate for 40 minutes at room temperature (RT). Wash the antennae in 0.4% PBST (PBS with 0.4% Triton X-100), 3x 10 minutes in each, keeping them in the same dish. Use yellow tips to remove and add PBST solution. Use 90 &#181;l of the wash solution each time. 
	NOTE: Do not place the dish into a shaker during immunohistochemistry. Before removing or adding the solution into the dish, bring all the antennae into the center of the dish using the needle and carefully add or remove the solution from the edge of the dish.</li>
	<li>Block the antennae with 90 &#181;l of 5% normal horse serum in 0.1% PBST for 20 minutes at RT.</li>
	<li>After removing the blocking solution, incubate the antennae with 90&#181;l of primary antibodies in 0.1% PBST containing 5% horse serum for 48 hr at 4 &#176;C in a moistened container as described previously8.</li>
	<li>Wash the antennae 6x 10 minutes in 0.4% PBST.</li>
	<li>Incubate the antennae with 90 &#181;l of secondary antibodies in 0.1% PBST containing 5% horse serum for 48 hr at 4 &#176;C. Wash 6x 10 minutes using 0.4% PBST.</li>
	<li>To mount the antennae, remove PBST from the culture dish as much as possible and gradually introduce two different concentrations of glycerol to the antennae. First add 40% glycerol to the dish for 1 to 2 minutes; then remove this and add 80% glycerol.</li>
	<li>Carefully extract the antennae (including 80% glycerol) from the culture dish using yellow tip and place them onto a slide. Gently place a coverslip on top and seal the coverslip edges with nail polish. The antennae are now ready to be imaged by fluorescence microscopy.</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>

The authors have nothing to disclose.

The dissection of the Drosophila antenna we describe is simple and easy to perform in a laboratory setting. To ensure a successful dissection, it is essential to utilize fine-edged scissors. While immunostaining the dissected antenna, it is important to incubate them in a moisture-filled container to avoid evaporation of the antibody solution. The dissected antenna has a tendency to float in the solution. Using 0.1% Triton in PBS during the fixation and blocking steps will facilitate submersion of the antenna in the solution and to ensure better staining. Using "glass bottom culture dish" could reduce the loss of antennae during immunostaining and ensure the small amounts (90 &#181;l) of antibody solution used in each experiment.
Neuronal-class diversification is a central feature of neurogenesis. This physiological process is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. Generation of a wide variety of ORNs with odorant receptor expression and axonal targeting is crucial to generating the neuronal diversity required for transmitting information from odor molecules to higher brain centers. Our whole mount antenna immunostaining protocol aids in furthering our understanding of the molecular mechanisms underlying ORN diversification.

The olfactory system is used to distinguish between an immense variety of odor molecules and subsequently to send the resulting information to the higher brain centers. This input is used to precisely control fundamental animal behaviors, such as feeding and mating1-6. As each olfactory neuron type is associated with a specific set of odors, the diversification of olfactory receptor neurons (ORN)s is essential for proper olfactory system function7.
Drosophila genetics enables us to perform single cell level investigation involving molecular mechanisms associated with ORN development and physiological function 8-16. Whole mount immunostaining of Drosophila antennae has enabled us to understand in greater detail the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s7. Herein we provide a comprehensive description of a simple method to achieve this.

<table><tbody><tr><td>Stemi DV4 dissection microscope</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Glass bottom culture dishes&amp;nbsp;</td><td>MatTek corporation</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Dissection scissor</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Rat anti-ELAV</td><td>Developmental Studies Hybridoma Bank</td><td>7E8A10</td><td>Dilution 1:200</td></tr><tr><td>Mouse anti-GFP</td><td>Invitrogen</td><td>A11122</td><td>Dilution 1:400</td></tr><tr><td>Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>711-225-152</td><td>Dilution 1:200</td></tr><tr><td>Donkey Anti-Rat IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>712-165-150</td><td>Dilution 1:200</td></tr></tbody></table>

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.

Ensuring that both the dissection and fixation are performed quickly is a key factor in achieving success with this protocol. Using fine scissors and forceps is also crucial. After immunostaining, fluorescent labeled antennae were examined under a confocal microscope. We normally take 1&micro;m sections using a 20x lens. We labeled Nba7 ORNs using Or49a-mCD8::GFP and counted the number of Nba ORNs in wild-type antenna. The mCD8-GFP reporter is cell membrane localized and so the expression seen in Figure 2 exhibits the cell membrane of OR49a ORNs expressing GFP. In Figure 2 shows Nba ORNs expression using anti GFP antibody and anti ELAV was used as a pan-neural marker. The average number of Nba ORNs per antenna is 20 (n=8).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51245/51245fig1highres.jpg" width="500" /> 
Figure 1: General overview of the dissection procedure. 
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51245/51245fig2highres.jpg" width="500" /> 
Figure 2: Projection of confocal Z-series of a successfully dissected antenna. To detect neurons anti-ELAV antibody was used as a pan-neural marker (A) and anti-GFP antibody to detect specific odorant receptor expression (B) and merged picture as shown in (C).

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K Views.  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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

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.

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

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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.

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

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Summary

Explore More Videos

Chapters in this video

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

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

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

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

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility