Odor–Taste Learning Assays in Drosophila Larvae

Abstract

The Drosophila larva is an emerging model for studies in behavioral neurogenetics because of its simplicity in terms of cell number. Despite this simplicity, basic features of neuronal organization and key behavior faculties are shared with adult flies and with mammals. Here, we describe a Pavlovian-type learning assay in fruit fly larvae. A group of larvae is sequentially exposed to specific odors in the presence or the absence of sugar, and then tested to determine whether they prefer the odor previously experienced with the reward. The protocol uses a two-group, reciprocal training design: One group of Drosophila larvae is exposed to n-amyl acetate (AM) with a sugar reward (+), then subsequently exposed to 1-octanol (OCT) with no reward (denoted AM+/OCT). The other group receives the reciprocal training (AM/OCT+). The two groups of larvae are then tested for their choices between AM and OCT. Relatively higher preferences for AM after AM+/OCT training than after AM/ OCT+ training reflect associative learning and are quantified by the learning index (LI). This method offers a robust, simple, cheap, and reasonably quick test for learning ability (an aversive version is available as well, using either high-concentration salt or quinine as punishment). With the concerted efforts of the Drosophila research community, we anticipate it will allow us to unravel the full circuitry underlying odor-taste learning on a single-cell level. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. ReagentsAgarose, 1% solution in distilled water Agarose, 1% solution, supplemented with 2 M fructose or sucrose Drosophila larvaeFlies are reared in culture bottles at 25˚C, with a relative atmospheric humidity of 60%-70% and with a 14/10-h light/dark cycle. Five-day-old larvae are used (90-120 h after egg laying); in particular, only larvae that are still in the food paste, and not larvae that may have begun to crawl up the side of the glass (known as the "wandering stage"), should be used for the learning assay.

Maggot learning and Synapsin function

“…After three such training cycles, larvae are tested for their choice between AM versus OCT (for half of the cases, the sequence of training trials is reversed: OCT/AM+ and AM/OCT+). From the difference in preference between the reciprocally trained groups the performance index is calculated to quantify associative learning (for details, see Gerber et al, 2010). (B)Associative function is reduced by ~50% in syn 97 mutant larvae relative to CS wild-type larvae; this effect is also seen in the w 1118 background.…”

Drosophila females trade off good nutrition with high quality oviposition sites when choosing foods

“…To evaluate the consequences of the egg-laying decisions of females on the cognitive abilities of larvae, we measured the learning performances of third instar larvae reared on three different breeding diets using a well-established reciprocal, differential conditioning assay for olfactory learning (Gerber et al, 2013). Fifteen groups of five females reared on a standard diet were transferred to culture tubes (55 ml) containing a high carbohydrate diet (P:C 1:16, P+C 180 g l −1 ), a high protein diet (P:C 8:1, P+C 180 g l −1…”

Cellular site and molecular mode of synapsin action in associative learning

“…Learning experiments follow standard methods (Scherer et al 2003;Neuser et al 2005; for a detailed protocol see Gerber et al 2010) (sketch in Fig. 1C), employing a two-odor, reciprocal conditioning paradigm, unless mentioned otherwise.…”