Olfactory Conditioning in the Third Instar Larvae of Drosophila melanogaster Using Heat Shock Reinforcement

Abstract

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.

A Low Concentration of Ethanol Impairs Learning but Not Motor and Sensory Behavior in Drosophila Larvae

“…The olfactory paradigm and conditioning protocol used in this study [32] were established for early third instar larvae.…”

“…The experimental set up was similar to a previous study [32]. In associative conditioning, a conditioned stimulus (CS) is paired with an unconditioned stimulus (US) to produce an altered or conditioned behavioral response to the CS.…”

“…Adult flies have long been used to study behavioral plasticity, but over the last few years, larval Drosophila have become valuable as a genetic model for the study of learning and memory [32]–[36]. With a powerful genetic toolbox and a simple nervous system that generates a variety of behaviors, larvae are an excellent choice for genetic analysis of neural plasticity.…”

“…We use heat-shock conditioning, a robust larval associative learning paradigm [32], to explore the effect of low pharmacologically relevant doses of ethanol on learning in Drosophila melanogaster larvae.…”

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

“…In addition, several variants of the paradigm were recently published that allow the investigation of associative visual learning in the larvae 23,36 (established in Gerber et al (2004)); and electric shock, light, heat, quinine or vibrations were also successfully implemented as aversive reinforcers for associative olfactory learning 9,17,[19][20][21][37][38][39][40] . Thus, a comprehensive set of experimental setups exists to analyze the behavioral, neuronal and molecular basis of learning and memory in Drosophila larvae ( Figure 5) 13,14,41,42 .…”