Name: functional evaluation of olfactory pathways in living xenopus tadpoles
Uploaded: 2018-12-11T13:00:00
Description: Watch this Scientific Journal Video about Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles at JoVE.com

This work was supported by grants from El Ministerio de Econom&#237;a y Competitividad (MINECO; SAF2015-63568-R) cofunded by the European Regional Development Fund (ERDF), by competitive research awards from the M. G. F. Fuortes Memorial Fellowship, the Stephen W. Kuffler Fellowship Fund, the Laura and Arthur Colwin Endowed Summer Research Fellowship Fund, the Fischbach Fellowship, and the Great Generation Fund of the Marine Biological Laboratory and the National Xenopus Resource RRID:SCR_013731 (Woods Hole, MA) where a portion of this work was conducted. We also thank CERCA Program/ Generalitat de Catalunya for institutional support. A.L. is a Serra H&#250;nter fellow.

All procedures were approved by the animal research ethics committee at University of Barcelona.
NOTE: X. tropicalis and X. laevis tadpoles are reared according to standard methods13,14. Tadpole water is prepared by adding commercial salts (see Table of Materials) to water obtained by reverse osmosis. Conductivity is adjusted to &#8764;700 &#181;S and &#8764;1,400 &#181;S for X. t...

<ol>
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This paper describes techniques that are useful to investigate the functionality of olfactory pathways in living Xenopus tadpoles. The current protocol is particularly useful for those laboratories that work, or have access to Xenopus; however, it is also interesting for those researchers studying the cellular and molecular bases of neuronal regeneration and repair. Results obtained in Xenopus can be combined with data gathered in other vertebrate models to identify conserved mechanisms. The me...

Xenopus tadpoles constitute an excellent animal model to study the normal function of the nervous system. Transparency, a fully sequenced genome1,2, and accessibility to surgical, electrophysiological and imaging techniques are unique properties of Xenopus larvae that allow investigating neuronal functions in vivo3. Some of the multiple experimental possibilities of this animal model are illustrated by the thorough studies performed on tadpole sensory and motor systems4,5,6. A particularly well-suited neuronal circuit to study many aspects of information processing at the level of synapses is the Xenopus tadpole olfactory system7. Firstly, its synaptic connectivity is well defined: olfactory receptor neurons (ORNs) project to the olfactory bulb and establish synaptic contacts with dendrites of mitral/tufted cells within glomeruli to generate odor maps. Secondly, its ORNs are continuously generated by neurogenesis throughout life to maintain the functionality of olfactory pathways8. And thirdly, because the olfactory system shows a great regenerative capability, Xenopus tadpoles are able to entirely reform their olfactory bulb after ablation9.
In this paper, we describe approaches that combine imaging of olfactory glomeruli in living tadpoles with behavioral experiments to study the functionality of olfactory pathways. The methods detailed here were used to study the functional recovery of glomerular connectivity in the olfactory bulb after olfactory nerve transection10. Data obtained in Xenopus tadpoles are representative of vertebrates since olfactory processing is evolutionary conserved.
The methods described are exemplified using X. tropicalis but they can easily be implemented in X. laevis. Despite the larger size of adult X. laevis, both species are remarkably similar during tadpole stages. The main differences reside at the genomic level. X. laevis displays poor genetic tractability, mostly determined by its allotetraploid genome and long generation time (approximately 1 year). In contrast, X. tropicalis is more amenable to genetic modifications due to its shorter generation time (5&#8211;8 months) and diploid genome. The representative experiments are illustrated for wild-type animals and three different transgenic lines: Hb9:GFP (X. tropicalis), NBT:GFP (X. tropicalis) and tubb2:GFP (X. laevis).
The methodologies outlined in the current work should be considered alongside the genetic progresses in the Xenopus field. The simplicity and easy implementation of the techniques presented makes them particularly useful for evaluating already described mutants11, as well as Xenopus lines generated by CRISPR-Cas9 technology12. We also describe a surgical procedure used to transect olfactory nerves that can be implemented in any laboratory having access to Xenopus tadpoles. The approaches used for evaluating presynaptic calcium responses and olfactory-guided behavior require specific equipment, albeit available at a moderate cost. Methodologies are presented in a simple form to promote their use in research groups and could set the bases of more complex assays either by implementing improvements or by the association to other techniques, i.e., histological or genetic approaches.

<table border="0" cellpadding="0" cellspacing="0" style="width:1133px;" width="1132">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Salts for aquariums (Instant Ocean Salt)</td>
			<td style="width:359px;">Tecniplast</td>
			<td style="width:143px;">XPSIO25R</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tweezers #5 (tip 0.025 x 0.005 mm)</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vannas Scissors (tip 0.015 x 0.015)</td>
			<td>World Precision Instruments</td>
			<td>501778</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Whatman qualitative filter paper</td>
			<td>Fisher Scientific</td>
			<td>WH3030917</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">X. laevis tubb2-GFP</td>
			<td>National Xenopus Resource (NXR), RRID:SCR_013731</td>
			<td>NXR_0.0035</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">X.tropicalis NBT-GFP</td>
			<td>European Xenopus Resource Center (EXRC) RRID:SCR_007164</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CellTracker CM-DiI</td>
			<td>ThermoFisher Scientific</td>
			<td>C-7001</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcium Green dextran, Potassium Salt, 10,000 MW, Anionic</td>
			<td>ThermoFisher Scientific</td>
			<td>C-3713</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Borosilicate capillaries for microinjection</td>
			<td>Sutter Instrument</td>
			<td>B100-75-10</td>
			<td>O.D.=1.0 mm., I.D.=0.75 mm.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microinjector</td>
			<td>Parker Instruments</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sylgard-184</td>
			<td>Sigma-Aldrich</td>
			<td>761028-5EA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microfil micropipettes</td>
			<td style="width:359px;">World Precision Instruments</td>
			<td>MF28G-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Upright microscope</td>
			<td>Zeiss</td>
			<td>AxioImager-A1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Master-8 stimulator</td>
			<td>A.M.P.I.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCD Camera</td>
			<td>Hamamatsu</td>
			<td>Image EM</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Solenoid valves</td>
			<td>Warner Instruments</td>
			<td>VC-6 Six Channel system</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dow Corning High Vacuum Grease</td>
			<td>VWR Scientific</td>
			<td>636082B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tubocurarine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T2379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCD Camera</td>
			<td>Zeiss</td>
			<td>MRC-5 Camera</td>
			<td>Controlled by Zen software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">camera lens</td>
			<td>Thorlabs</td>
			<td>MVL8ML3</td>
			<td>There are multiple possibilities that should be adapted to the camera model used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epoxy resin</td>
			<td>RS Components</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manifold</td>
			<td>Warner Instruments</td>
			<td>MP-6 perfusion manifold</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator for local delivery of solutions</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini magnetic clamps</td>
			<td>Warner Instruments</td>
			<td>MAG-7, MAG-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing</td>
			<td>Warner Instruments</td>
			<td>64-0755</td>
			<td>O.D.=1.57 mm., I.D.=1.14 mm.</td>
		</tr>
	</tbody>
</table>

functional evaluation of olfactory pathways in living xenopus tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

In this paper, we present a combination of two complementary approaches to perform in vivo study of the functionality of the Xenopus tadpole olfactory system: i) a method for imaging presynaptic Ca2+ changes in the glomeruli of living tadpoles using a fluorescent calcium indicator, and ii) an odor guided behavioral assay that can be used to investigate the response to specific waterborne odorants. Since these approaches have been employed to evaluate the recov...

Watch this Scientific Journal Video about Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles at JoVE.com

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Research

JoVE Journal

Neuroscience

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 ...

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Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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

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An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

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The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...