GoKawiilDrosophila strains and culture
Flies (D. melanogaster) were raised on standard medium (inactivated yeast 6% (w/v); corn flour 6.66% (w/v); agar 0.9% (w/v); methyl-4hydroxybenzoate 22 mM) under a 12 h–12 h light–dark cycle at 18 °C with 60% humidity (unless mentioned otherwise). All of the experiments were performed on young (aged <5 days) adult flies. For behaviour experiments, groups of mixed-sex flies were used unless indicated otherwise. For imaging experiments involving surgery, female flies were used because of their larger size. All flies obtained from libraries or received after the injection of transgenes were outcrossed for five generations to a reference strain. In general, this reference line carried the w1118 mutation in an otherwise Canton-S genetic background; as an exception, and because TRiP RNAi transgenes are labelled by a y+ marker, lines from this collection were outcrossed to a y1w67c23 strain in an otherwise Canton-S background. To restrict UAS/GAL4-mediated expression to the adult stage, we used the TARGET system involving the ubiquitous expression of the thermosensitive GAL4 inhibitor under a tubulin promoter (tub-Gal80ts)29. In general, for crosses involving binary expression control systems (GAL4/UAS and/or LexA/LexAop), female flies carrying the driver transgene(s) were crossed to male flies carrying the effector transgene(s). A list of all of the single-transgene strains used in this study is provided in Supplementary Table 9. When needed, fly lines carrying combinations of multiple transgenes were obtained from these lines through routine crossing schemes.
Classical aversive or appetitive olfactory conditioning
To induce RNAi expression using the TARGET system, adult flies (aged 0 to 2 days) were kept at 30.5 °C for 3 days before conditioning. In the case of appetitive conditioning, flies were transferred to starvation vials (containing only a mineral-water-soaked cotton disk) for the last 16 h of the induction time. For experiments that did not involve thermal induction of transgene expression, experimental flies (aged 0-3 days) were transferred to fresh bottles containing standard medium 24 h before conditioning in the case of aversive conditioning experiments. For appetitive conditioning, flies (aged 0–2 days) were transferred to fresh food vials 1 day before being transferred to starvation vials for 21 h at 25 °C.
The aversive behaviour experiments, including sample sizes, were conducted similarly to other studies from our research group. Groups of 20–50 flies were subjected to one of the following olfactory conditioning protocols: a single cycle (1× training; duration of around 4 min), five consecutive associative training cycles (5× massed training; duration of around 20 min) or five associative cycles spaced by 15 min intertrial intervals (5× spaced training; duration of around 1 h 30 min). Non-associative control protocols (unpaired protocols) were also used for imaging experiments. Conditioning was performed using previously described barrel-type machines that allow parallel training of up to six groups. Throughout the conditioning protocol, each barrel was plugged into a constant air flow at 2 l min−1. For a single cycle of associative training, flies were first exposed to an odorant (the CS+) for 1 min while 12 pulses of 5-s-long 60 V electric shocks were delivered; flies were then exposed 45 s later to a second odorant without shocks (the CS−) for 1 min. The odorants 3-octanol and 4-methylcyclohexanol, diluted in paraffin oil to a final concentration of 2.79 × 10−1 g l−1, were alternately used as conditioned stimuli. In all of the behaviour experiments, two conditioning protocols were conducted sequentially on two batches of genotypically identical flies: a first set of flies was conditioned with 3-octanol as CS+ and 4-methylcyclohexanol as CS−, and a second reciprocal set with 4-methylcyclohexanol as CS+ and 3-octanol as CS−. This enabled us to balance for potential systematic choice bias during memory retrieval test (see below), as is commonly done in Drosophila memory experiments. During unpaired conditionings, the odour and shock stimuli were delivered separately in time, with the onset of electric shock delivery occurring 3 min and terminating 2 min before the first odorant.
The appetitive behaviour experiments, including the sample sizes, were conducted similarly to other studies from our research group. During appetitive conditioning, flies were first exposed to an odorant (the CS+) for 1 min paired with a dried sugar reward, followed 45 s later by a second odorant (the CS−) presented without reward for 1 min, as described previously46. The odorants were the same, and were used at the same concentration, as for the aversive conditioning experiments. Plexiglas tubes covered with dried sugar were prepared the day before conditioning: a 1.5 M sucrose solution in mineral water was spread on the tube surface and tubes were placed in front of a ventilator overnight at room temperature.
Test of memory retrieval
For aversive memory behaviour experiments, unless indicated otherwise in the figures, flies were kept on standard medium between conditioning and the memory test, at either 25 °C for flies tested 3 h after 1× training, or at 18 °C for flies tested 24 h after training. After 1× training, to assess separately the two components of memory formed after 1× training (middle-term memory, which is anaesthesia sensitive, and anaesthesia-resistant memory), a subset of the trained groups were subjected to a 4 °C cold treatment for 2 min, 2 h after training (that is, 1 h before the memory test). For appetitive memory behaviour experiments, flies were kept in starvation vials between conditioning and the memory test. The memory test was performed in a T-maze apparatus, typically 3 h after single-cycle training or 24 h after massed or spaced training. Each arm of the T-maze was connected to a bottle containing 3-octanol or 4-methylcyclohexanol, diluted in paraffin oil to a final concentration identical to the one used for conditioning. Flies were given 1 min in complete darkness to choose between either arm of the T-maze. A score was calculated as the number of flies in the CS− arm of the T-maze minus the number of flies in the CS+ arm, divided by the total number of flies in both arms. To balance for potential systematic choice bias due to a preference for either side of the maze, we combined the performance of flies conditioned with either 3-octanol or 4-methylcyclohexanol as the CS+ condition. Thus, a single performance index value reported on graphs is the average of two scores obtained from two groups of genotypically identical flies conditioned sequentially using either odorant (3-octanol or 4-methylcyclohexanol) as the CS+ condition. In particular, for cases that required counting a subset of the assayed flies after the memory test (for example, disaggregated evaluation of female and male flies, or the presence of a balancer chromosome in a parental line), scores involving fewer than six flies in total were discarded to avoid giving disproportionate statistical importance to a small number of flies. The indicated n is the number of independent performance index values for each genotype.
Innate shock avoidance assessment
Shock-response tests were performed at 25 °C by placing flies in two connected chambers identical to those used for olfactory conditioning. Electric shocks were delivered in only one of the compartments. Flies were given 1 min to move freely in these compartments, after which they were trapped, collected and counted. The compartment where the electric shocks were delivered was alternated between two consecutive groups. Shock avoidance was calculated as for the memory performance.
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