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.
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Method Article
Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles
* These authors contributed equally
In This Article
Summary
Abstract
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.
Introduction
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....
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Protocol
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 ∼700 µS and ∼1,400 µS for X. tropicalis and X. laevis tadpoles, respectively. Larvae can be obtained either by natural mating or in vitro fertilization14. Embryos a....
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Results
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.......
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Discussion
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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from El Ministerio de Economía 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 Húnter fello....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Salts for aquariums (Instant Ocean Salt) | Tecniplast | XPSIO25R | |
| Tricaine (Ethyl 3-aminobenzoate methanesulfonate) | Sigma-Aldrich | E10521 | |
| Tweezers #5 (tip 0.025 x 0.005 mm) | World Precision Instruments | 501985 | |
| Vannas Scissors (tip 0.015 x 0.015) | World Precision Instruments | 501778 | |
| Whatman qualitative filter paper | Fisher Scientific | WH3030917 | |
| X. laevis tubb2-GFP | National Xenopus Resource (NXR), RRID:SCR_013731 | NXR_0.0035 | |
| X.tropicalis NBT-GFP | European Xenopus Resource Center (EXRC) RRID:SCR_007164 | ||
| CellTracker CM-DiI | ThermoFisher Scientific | C-7001 | |
| Calcium Green dextran, Potassium Salt, 10,000 MW, Anionic | ThermoFisher Scientific | C-3713 | |
| Borosilicate capillaries for microinjection | Sutter Instrument | B100-75-10 | O.D.=1.0 mm., I.D.=0.75 mm. |
| Puller | Sutter Instrument | P-97 | |
| Microinjector | Parker Instruments | Picospritzer III | |
| Sylgard-184 | Sigma-Aldrich | 761028-5EA | |
| Microfil micropipettes | World Precision Instruments | MF28G-5 | |
| Upright microscope | Zeiss | AxioImager-A1 | |
| Master-8 stimulator | A.M.P.I. | ||
| CCD Camera | Hamamatsu | Image EM | |
| Solenoid valves | Warner Instruments | VC-6 Six Channel system | |
| Dow Corning High Vacuum Grease | VWR Scientific | 636082B | |
| Tubocurarine hydrochloride | Sigma-Aldrich | T2379 | |
| CCD Camera | Zeiss | MRC-5 Camera | Controlled by Zen software |
| camera lens | Thorlabs | MVL8ML3 | There are multiple possibilities that should be adapted to the camera model used |
| Epoxy resin | RS Components | ||
| Manifold | Warner Instruments | MP-6 perfusion manifold | |
| Micromanipulator for local delivery of solutions | Narishige | MN-153 | |
| Mini magnetic clamps | Warner Instruments | MAG-7, MAG-6 | |
| Polyethylene tubing | Warner Instruments | 64-0755 | O.D.=1.57 mm., I.D.=1.14 mm. |
References
- Hellsten, U., et al. The genome of the Western clawed frog Xenopus tropicalis. Science. 328 (5978), 633-636 (2010).
- Session, A. M., et al. Genome evolution in the allotetraploid frog Xenopus laevis. Nature. 538 (7625), 336-343 (2016).
- Zhang, L. I., Tao, H. ....
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