DOI: 10.5061/dryad.v9s4mw6t1
title: Fruit flies can learn non-elemental olfactory discriminations

description: Associative learning allows animals to establish links between stimuli
 based on their concomitance. In the case of Pavlovian conditioning, a
 single stimulus A (the conditional stimulus, CS) is reinforced
 unambiguously with an unconditional stimulus (US) eliciting an innate
 response. This conditioning constitutes an ‘elemental’ association
 enabling to elicit a learnt response from A+ without US presentation after
 learning. However, associative learning may involve a ‘complex’ CS
 composed of several components. In that case, the compound may predict a
 different outcome than the components taken separately, leading to an
 ambiguity and requiring the animal to perform a so-called ‘non-elemental’
 discrimination. Here we focus on such a non-elemental task, the negative
 patterning (NP) problem, and provide the first evidence of NP solving in
 Drosophila. We show that Drosophila learn to discriminate a simple
 component (A or B) associated to electric shocks (+) from an odour mixture
 composed either partly (called ‘feature-negative discrimination’ A+ vs.
 AB-) or entirely (called ‘NP’ A+B+ vs. AB-) of the shock associated
 components. Furthermore, we show that conditioning repetition results in a
 transition from an elemental to a configural representation of the mixture
 required to solve the NP task, highlighting the cognitive flexibility of
 Drosophila.
description: <strong>Fly rearing: </strong>The wild-type line used
 in this study was a <i>Canton-Special
 </i>(<i>Canton-S</i>) strain. Flies were raised on
 standard medium at 25°c, ~60% humidity and a 12h/12h light/dark cycle. The
 flies were kept in 36x82mm plastic tubes containing ~20mL of
 medium. <strong>Olfactory conditioning:
 </strong>Odours were diluted in a bottle of mineral oil. Odours used
 were 3-Octanol (termed ‘A’ for the sake of simplicity, 2.27mM) and
 4-Methylcyclohexanol (‘B’, 2.62mM) (figure 1 and figure 2). Benzaldehyde
 (‘C’, 1.89 mM) was used as a novel odour in some conditions (figure 2).
 IsoamylAcetate (‘D’) and EthylButyrate (‘E’) were used as alternative
 odours (electronic supplementary material, figure S2). Odours are prepared
 at the same concentration whether as components (A, B, D or E) or as
 mixtures (AB or DE), which is essential for studying non-elementary
 learning. In the case of mixtures, odours were diluted together in the
 same bottle of mineral oil. All odours along with the solvent were from
 Sigma Aldrich (France). The US consisted of twelve 1.5s pulses of 60V
 electric shock every 5s delivered through a metallic grid. Each experiment
 included two groups of ~30 flies (2 – 4 days old) and was performed using
 a semi-automated device based on a previous work. In a T-Maze, two main
 phenomena drive the preference of flies toward a compartment or another:
 on the one hand, the learnt information about the stimuli acquired during
 conditioning and on the other hand, the fact that odours are rarely
 completely neutral; at the concentrations used in our work, they are in
 fact repulsive to naïve flies. When two odorants are opposed in the
 absence of punishment in the T-maze, repulsion balances; yet, if one
 odorant is opposed to a compound more repulsive, a bias towards the less
 repulsive stimulus is visible. To disentangle learning from non-learning
 behavioural components, one of the groups experienced an explicit pairing
 of CS and US (‘paired group’) while the other group experienced both
 stimuli unpaired to prevent their association (‘unpaired
 group’). <strong>Training: </strong>Each
 training trial consisted of 90 s of acclimatisation, after which flies
 were subjected to their respective conditioning protocol. Each of the two
 odours (CS) was presented once for 1min with an intertrial interval of
 1min. For the paired group (figure 1<i>a</i>), one of the
 olfactory stimulus (CS<sup>+</sup>) was paired with the US,
 while the other stimulus (CS<sup>-</sup>) remained unpunished.
 In the unpaired group, flies were exposed to 1min of either shocks or
 odours, separated by an interval of 1min. This sequence formed one
 conditioning cycle. Flies were subjected to one of
 three training protocols (figure 1<i>b</i>): a differential
 conditioning (DC) in which they had to learn to discriminate a punished
 from a non-punished odour (A<sup>+</sup> vs.
 B<sup>-</sup>), a NF discrimination in which they had to learn
 to discriminate a punished odour from a non-punished odour compound
 (A<sup>+</sup> vs. AB<sup>-</sup>) and a NP
 discrimination in which they had to learn to discriminate two punished
 odours from a non-punished odour compound (A<sup>+</sup>,
 B<sup>+</sup> vs. AB<sup>-</sup>). 3-Octanol was
 always used as the CS+ for DC and NF protocols (with respectively
 4-Methylcyclohexanol and 3-Octanol+4-Methylcyclohexanol as CS-). For NP
 protocol, both 3-Octanol and 4-Methylcyclohexanol were used as CS+ when
 presented alone and as CS- when presented as a compound. Thus, flies
 subjected to DC training faced a pure elemental discrimination. On the
 contrary, flies trained in the NP protocol could only solve the problem if
 they adopted a non-elemental strategy. Finally, flies subjected to NF
 training could solve the problem using either an elemental or a
 non-elemental strategy. For each protocol, training consisted of either
 one or five cycles, which allowed studying if the amount of experience
 gathered by flies promoted a particular discriminations
 strategy. <strong>Test: </strong>After
 training, flies were transferred to a T-maze where they could choose
 between the CS<sup>+</sup> and the CS<sup>-</sup>
 in the absence of shock during 1min. Flies from paired and unpaired
 protocols were sequentially tested. At the end of the test, flies in each
 arm of the T-maze were counted. If paired flies learned the
 discrimination, they should be mostly located in the CS- arm, the arm
 presenting the odour stimulus that was not associated to the shocks during
 the training. A Performance Index (PI) was calculated as: (Number of flies
 in the CS<sup>-</sup> arm – Number of flies in the
 CS<sup>+</sup> arm) / total Number of flies. To control for
 any experimental bias, each replicate consisted of a ‘paired-group’ PI
 (reflecting associative learning + bias) from which an ‘unpaired-group’ PI
 (reflecting bias only) was substracted. 
description: Please note that no data sheet is available for the electronic
 supplementary material figure S1 because we used the same data as in Fig1,
 but we represented all scores (Paired/Unpaired/Corrected index) instead of
 only the corrected index to illustrate how comparing Paired and Unpaired
 scores allows for accurate assocative learning quantification. We also
 only plotted data after 5 conditioning trials in the figure S1.