Appetitive Associative Olfactory Learning in <em>Drosophila</em> Larvae

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 traces [1][2][3][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 vertebrates 5,6 . 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][8][9][10] . Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar [11][12][13][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 groups 9 . Video LinkThe video component of this article can be found at http://www.jove.com/video/4334/ Protocol 1. Preparation 1. 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 3 rd instar stage if raised at 25 °C and can now be used for the behavioral experiment. However, one has to ensure to only take larvae that are still in the food and not the larvae from the side of the vial. These larvae have alread...

Neuropeptide F neurons modulate sugar reward during associative olfactory learning of Drosophila larvae

“…Appetitive olfactory learning was tested by using standardized, previously described assays (Scherer et al, ; Neuser et al, ; Schipanski et al, ; Pauls et al, ; Rohwedder et al, ; Apostolopoulou et al, ). In detail, all learning experiments were conducted on assay plates filled with a thin layer of agarose solution containing either pure 2.5% agarose or 2.5% agarose plus fructose at concentrations of 2M or 0.02M.…”

“…AM), the BA side (No. BA), and in a 1‐cm neutral zone in between (for further details a video is available in Apostolopoulou et al, ). Due to the stressful heat and the light treatment applied in experiments for artificial thermal and light activation of dNPF neurons we reduced the training intervals to 2.5 minutes per plate in these experiments.…”

“…For olfactory acuity test, 2.5% agarose solution (Sigma‐Aldrich) was boiled in a microwave oven and filled as a thin layer into Petri dishes (85 mm diameter, Sarstedt) (Apostolopoulou et al, ). After cooling, closed Petri dishes were kept at room temperature and were used on the same day.…”

“…Drosophila larvae are able to form appetitive olfactory memories following odorant sugar reward pairing and a standardized behavioral paradigm exists for its analysis (Scherer et al, ; Neuser et al, ; Gerber and Stocker, ; Schipanski et al, ; Rohwedder et al, ; Apostolopoulou et al, ). Similar to other insects, it was shown that after appetitive olfactory conditioning a memory trace is established in Kenyon cells of the mushroom body (MB), a second‐order “cortical” brain region (Honjo and Furukubo‐Tokunaga, ; Pauls et al, ; Tomer et al, ; Michels et al, ).…”

Reward signaling in a recurrent circuit of dopaminergic neurons and Kenyon cells

“…Appetitive olfactory learning was tested using standardized, previously described assays 5,59 . Learning experiments were performed on plates filled by a thin layer of pure 1.5% agarose solution (Roth, 9012-36-6).…”