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Summary
Abstract
Protocol
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Neurociencias

Appetitive Associative Olfactory Learning in Drosophila Larvae

Published: February 18, 2013
doi:
10.3791/4334
, , , ,
1Department of Biology,University of Konstanz, 2Department of Biology,University of Fribourg

Summary

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Abstract

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.

Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.

Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning – presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Protocol

1. Preparation Drosophila wild-type larvae are raised at 25 °C and 60%-80% humidity in a 14/10 light/dark cycle. For controlling the exact age of the larvae always 20 females are put with 10 males into one vial (6 cm height and 2.5 cm diameter) that includes about 6 ml of standard fly food. Flies are allowed to lay eggs for 12 hr and are transferred to a new vial on the second day. 5-6 days after egg laying larvae reach the feeding 3rd instar stage if raised at 25 °C and can now be…

Representative Results

Figure 1A shows an overview of the experimental procedures for larval olfactory associative learning. By pairing one of the two presented odors with a sugar reward larvae acquire the behavior potential to express an attractive response towards the rewarded odor in comparison to the unrewarded odor. Two groups of larvae are always trained by either pairing the reinforcer with the odor OCT or AM. The performance index (PI) measures the associative function as the difference in preference between the…

Discussion

The described setup in Drosophila larvae allows for the investigation of associative olfactory learning within a comparably elementary brain. The approach is simple, cheap, easy to establish in a lab and does not require high-tech equipment9. We present a version of the experiment, to study appetitive associative learning reinforced by fructose reward11. The described setup is based on a series of parametrical studies that comprehensively investigated variations in the number of trai…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

We especially want to thank the members of the Gerber lab for technical instructions on their experimental setup and comments on the manuscript. We also thank Lyubov Pankevych for fly care and maintenance of the wild type CantonS stock. This work is supported by the DFG grant TH1584/1-1, the SNF grant 31003A_132812/1 and the Zukunftskolleg of the University of Konstanz (all to AST).

Materials

Name of the reagent Company Catalogue number CAS number
Fructose Sigma 47740 57-48-7
NaCl Fluka 71350 7647-14-5
Agarose Sigma A5093 9012-36-6
1-octanol Sigma 12012 111-87-5
Amylacetate Sigma 46022 628-63-7
Paraffin oil Sigma 18512 8012-95-1
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Tags

AppetitiveAssociativeOlfactory LearningDrosophila LarvaeGenetic InterferenceNeuronal ChangesMolecular ChangesMemory TracesModel OrganismsSimpleExperimentally AccessibleBehavioral AssaysTransgenic TechniquesNervous SystemOdorsGustatory ReinforcementSugar
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Apostolopoulou, A. A., Widmann, A., Rohwedder, A., Pfitzenmaier, J. E., Thum, A. S. Appetitive Associative Olfactory Learning in Drosophila Larvae. J. Vis. Exp. (72), e4334, doi:10.3791/4334 (2013).
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