Name: whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna
Uploaded: 2014-05-04T14:00:00
Description: Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

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>An...

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

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

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 exhi...

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 ...

Research

JoVE Journal

Neuroscience

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

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

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

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

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 ...

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

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

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

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

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

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

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

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 ...

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

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

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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