Learning performance of normal and mutant Drosophila after repeated conditioning trials with discrete stimuli

Abstract

A new olfactory conditioning procedure is described using short training trials with discrete presentation of conditioned stimuli (CS) and unconditioned stimuli (US). A short odor presentation along with a single-shock stimulus produced modest but reliable and reproducible learning. Multiple trials presented sequentially improved performance with increasing trial number. Trial spacing had a significant impact on performance. Two trials presented with a short intertrial interval (ITI) produced no improvement over a single trial; two trials with a 15 min ITI significantly boosted performance. This effect required two associative trials, because substituting one of the trials with the CS alone, US alone, or an unpaired CS-US failed to boost performance. The increase in initial performance with two trials decayed within 15 min after training. Thus, the effect is short-lived. The utility of using a battery of tests, including a single short trial, two massed trials, and two spaced trials, to investigate parameters of memory formation in several mutants was demonstrated.

Fig. 1.

Performance with altered CS and US duration.a, Performance decrement with decreased CS exposure and US number. b, Performance of flies in a retest that had made a previous correct or incorrect choice. c, Effects of increasing electric shock duration with a 10 sec CS+ exposure. No significant effect was observed, although there was a trend toward high performance with longer US exposure. Statistics are as follows.a, One-factor ANOVA revealed a significant effect (F_(4,25) = 51.1; p< 0.01; n = 6 in each group) between groups. Fisher post hoc comparisons revealed significant differences between all groups. A two-tailed, one-samplet test with population mean of 0 revealed the 10 sec, 1 shock condition produced significant conditioning (t₍₅₎ = 11.1; p < 0.01). b, One-factor ANOVA was not significant (F_(2,24) = 1.07; NS;n = 9 for all groups). Mean ± SEM; flies in first test, 38 ± 3; correct flies in retest, 27 ± 4; incorrect flies in retest, 31 ± 7. c, One-factor ANOVA revealed no significant effect (F_(2,15) = 1.9; NS;n = 6 per group). Mean ± SEM; 1.25 sec, 28 ± 5; 5 sec, 36 ± 4; 10 sec, 41 ± 5.

Fig. 2.

Effect of intertrial interval. Performance was measured after a single trial or after two trials separated by the time indicated. Optimal performance occurred with ITIs of 10 and 15 min. Statistics are as follows: one-factor ANOVA revealed a significant effect of ITI (F_(8,45) = 4.7;p < 0.01; n = 6 per group). Fisher post hoc comparisons revealed that performance with a 15 min ITI was significantly elevated over all other groups, except after training at a 10 min ITI.

Fig. 3.

Effect of multiple trials at two different ITIs. a, Different groups were trained with 1–20 trials at either a 30 sec or 15 min ITI. Two trials at a 30 sec ITI did not significantly increase performance over one trial, but subsequent trials at this ITI elevated performance. Two trials at a 15 min ITI, however, produced significantly higher performance than a single trial or two trials at a 30 sec ITI. A further significant jump in performance was observed with groups given seven trials at a 15 min ITI. b, Effect of training session length. Three groups were trained, two receiving the same number of training trials, and two receiving the same total training session time. Trial number was the important factor in elevating performance. c, Performance after presenting a subset of seven, 15 min spaced training trials. Each group was trained concurrently over the time required for seven spaced trials, but the different groups received only the training trials indicated. Seven-trial training produced robust conditioning. The performance increase observed between five and seven spaced training trials (Fig. 3a) was not reproduced by presenting only trials 1 and 7; 1, 2, and 7; or 6 and 7. d,e, Effects of cycloheximide treatment on flies given two or seven spaced training trials. Flies were fed 35 mmcyclohexmide in 4% sucrose for 12 hr before training. Different groups then received single trial, two trials 30 sec ITI, two trials 15 min ITI, 5 trials 30 sec ITI, 5 trials 15 min ITI, 7 trials 30 sec ITI, and 7 trials 15 min ITI. The performance enhancement of spaced training with two trials (d) and spaced training with seven trials (e) was reproduced. The enhanced performance was independent of cyclohexmide treatment. Statistics are as follows. a, One-factor ANOVA revealed a significant effect of trial number at both ITIs: 30 sec ITI,F_(7,45) = 11.0; p< 0.01; n = 6–8 per group; 15 min ITI,F_(7,45) = 23.5; p< 0.01; n = 6–8 per group. The ITI affected performance (two-factor ANOVA; effect of ITI,F_(1,78) = 41.5; p< 0.01; effect of trials, F_(6,78) = 16.0; p < 0.01; interaction of ITI × trials,F_(6,78) = 3.0; p < 0.02. Post hoc comparisons showed significant increases in performance at a 30 sec ITI between two and three trials, and between three and 10 trials. Similar comparisons for groups trained with a 15 min ITI showed significant increases between one and two trials, and between two and seven trials. b, One-factor ANOVA showed that all three groups were different (F_(2,15) = 31.7; p< 0.01; n = 6 per group). Mean ± SEM; 5 trials 15 min ITI, 60 ± 3; 10 trials 15 min ITI, 81 ± 2; 5 trials 30 min ITI, 41 ± 5. c, There was an overall effect of training condition (F_(4,29) = 33.1; p < 0.01). Fisher post hocrevealed significant differences between all groups and the group receiving all seven trials. Mean ± SEM; trials 1–7, 82.0 ± 3.2; trials 1 and 7, 33.7 ± 3.0; trials 1, 2, and 7, 49.8 ± 3.6; trial 7, 32.2 ± 2.1; trials 6 and 7, 51.0 ± 4.9;n = 6 per group. d, There was a significant effect of training (F_(2,30)= 21.3; p < 0.01; n = 6 per training condition–treatment). There was no significant effect of cyclohexmide (CXM) treatment (F_(1,30) = 0.2; NS;n = 6 per training condition–treatment) and no significant interaction between training condition and cyclohexmide treatment (F_(2,30) = 0.9; NS;n = 6 per training condition–treatment). Fisherpost hoc revealed significant differences between two trials, 15 min ITI conditions (with or without cyclohexmide) and both other training conditions. e, There was a significant effect of training (F_(1,40) = 35.6;p < 0.01; n = 6 per training condition–treatment) and ITI (F_(1,40)= 53.4; p < 0.01; n = 6 per training condition–treatment), and a significant interaction between training trials and ITI (F_(1,40) = 27.3; p < 0.01; n = 6 per training condition–treatment). There was no significant effect of cyclohexmide treatment (F_(1,40) = 0.9; NS; n = 6 per training condition–treatment) nor significant interactions of cyclohexmide treatment with training trials (F_(1,40) = 0.4; NS;n = 6 per training condition–treatment) or ITI (F_(1,40) = 0.1; NS;n = 6 per training condition–treatment). Fisherpost hoc revealed significant differences between seven trials, 15 min ITI conditions (with or without cyclohexmide) and all other groups.

Fig. 4.

Effects of apparatus exposure on two-trial performance. a, An experimental group received two massed training trials after exposure to the apparatus for 17 min to make the total exposure time equivalent to the two trials, 15 min ITI group. b, An experimental group received two massed training trials before an additional 17 min exposure to the apparatus before the test. Statistics are as follows. a, Training after time in apparatus, F_(3,20) = 9.1;p < 0.01; n = 6 per group.b, Training before time in apparatus,F_(3,20) = 4.8; p < 0.01; n = 6 per group. Post hoccomparisons showed that the performance of all groups trained at a 30 sec ITI was significantly less than those trained at a 15 min ITI and was not different from those groups that received a single trial in training.

Fig. 5.

Effects of air, US alone, CS alone, or CS–US unpaired on two-trial spaced training. a, Two-trial conditioning with a 15 min ITI produced performance significantly elevated over groups conditioned with a single trial. Substitution of the first (a) or second (b) CS–US pairing with air, US alone, or CS alone produced performance equivalent to a single conditioning trial. Unpairing the CS and US on the first (a) or second (b) trial inhibited the association. Statistics are as follows.a, There was an overall effect of training (F_(5,30) = 9.1; p< 0.01; n = 6 per group). Fisher post hoc revealed no differences between the single-trial group and the groups given air, US alone, and CS alone. Significant differences were observed between the single-trial group and the CS–US paired and CS–US unpaired groups. b, There was an overall effect of training (F_(5,30) = 10.2;p < 0.01; n = 6 per group). Fisher post hoc revealed no differences between the single-trial group and the groups given air, US alone, and CS alone. Significant differences were observed between the single-trial group and the CS–US paired and CS–US unpaired groups.

Fig. 6.

Memory after training. Performance evaluated at 3 min, 15 min, 1 hr, and 3 hr after single trial, two trials massed, or two trials spaced conditioning. The spaced training produces higher performance immediately after training (3 min), but the effect wanes rapidly, disappearing by 15 min after training. Statistics are as follows. There was a significant decrease in the level of performance with later retention intervals (F_(3,60)= 8.9; p < 0.01; n = 6 per training condition–retention interval). There was an overall significant effect of the training procedure (F_(2,60) = 5.3; p< 0.01), but no significant interaction between training procedure and retention interval (F_(6,60) = 0.5; NS). Data analyzed from each retention interval showed a significant effect of spaced training only at 3 min (F_(2,17) = 5.2; p< 0.02; 15 min, F_(2,17) = 1.0; NS; 1 hr, F_(2,17) = 1.9; NS; 3 hr,F_(2,15) = 0.6; NS).

Fig. 7.

Performance of memory mutants after a single trial, two massed trials, and two spaced trials. Training of thery control produced spaced effects similar to those observed in Figures 3-5, with enhanced performance when given two spaced trials. All mutants performed poorly after any training condition with differences in how the three mutants respond to the different training conditions. Therut²⁰⁸⁰ mutant showed a significant effect from spaced training. Vol¹ andVol² did not, although there was a trend in this direction for Vol¹. Naive performance was noted with Vol²given a single trial. Statistics are as follows. There was a significant effect of training and genotype (training,F_(2,60) = 3.3; p < 0.05; genotype, F_(3,60) = 12.3;p < 0.01; n = 6). There was no significant interaction between training and strain (F_(6,60) = 1.5; NS), but inspection of the graph shows that there may be differences in how the strains reacted to the training procedures. Post hoc comparisons showed that the performance of the three mutants was lower overall than that of ry and that the performance ofVol² was lower than that ofVol¹ orrut²⁰⁸⁰. When the data from each strain were analyzed separately, ry andrut²⁰⁸⁰ showed a significant effect of the training condition (ry,F_(2,15) = 4.0; p < .05; rut²⁰⁸⁰,F_(2,17) = 4.1; p < 0.05), whereas Vol¹ andVol² did not (Vol¹,F_(2,17) = 1.1; NS;Vol²,F_(2,17) = 0.6; NS).

Fig. 8.

Behavioral rescue of spaced training performance. There was no effect of a 15 min HS on the performance ofry flies after training with a single trial, two massed trials, and two spaced trials. Vol²flies perform poorly after training; HS had no effect on performance.Vol²(hspVol-s) flies performed like Vol² mutants in the absence of HS but performed like ry controls after HS. Statistics are as follows. Three-factor ANOVA revealed an effect of genotype (F_(2,90) = 53.3;p < 0.01), training procedure (F_(2,90) = 24.6; p< 0.01), and HS condition (F_(1,90) = 13.7; p < 0.01); n = 6 for all groups. There was a significant interaction of genotype and training procedure (F_(4,90) = 3.4;p < 0.05) and genotype and HS condition (F_(2,90) = 17.5; p< 0.01). Fisher post hoc revealed that the single-trial groups and the two massed trial groups were not different from one another but were both significantly different from the two spaced trial groups for ry (no HS), ry (HS), andVol²(hspVol-s) (HS). The single-trial groups were different from the two-trial massed and two-trial spaced groups for Vol² (no HS) and Vol² (HS). For the genotypes and training conditions [Vol² no HS, Vol² HS, andVol²(hspVol-2) no HS], one-sample, one-tailed t tests (population mean of 0) showed significant performance only for theVol²(hspVol-2) no HS group (t₍₅₎ = 2.068;p < 0.05). Neither theVol² no HS nor Vol² HS groups demonstrated any significant learning.

Fig. 9.

Dominance–recessivity after short program training. Flies of the genotypes listed were trained with a single trial, two massed trials, and two spaced trials. Immediate performance after training was measured. Vol¹ andVol² both exhibited deficits as before. Vol¹ was completely recessive after this training, because the performance ofVol¹/+ was identical to the control. Vol² was partially dominant for single-trial training, becauseVol²/+ performance after this training was elevated overVol² homozygotes but was not at wild-type levels. The Vol² phenotype after two massed trials or two spaced trials, however, was completely dominant, because the performance ofVol²/+ with this training was identical to Vol²homozygotes. The partially dominant phenotype (single trial) and completely dominant phenotype (two trials, massed or spaced) ofVol³ observed inVol³/+ flies is accounted for by the loss of the Vol-s transcript, given the behavioral similarities withVol²/+ andVol¹/Vol²flies. Conditioning after a single trial is completely dependent on the presence of at least one copy of the Vol-s transcription unit (behavior ofVol³/Vol²and Vol² homozygotes). Statistics are as follows: two-factor ANOVA revealed an effect of genotype (F_(7,120) = 28.0; p< 0.01), training procedure (F_(2,120)= 16.2; p < 0.01), and a significant interaction (F_(14,120) = 3.7; p< 0.01); n = 6 for all groups. Fisher post hoc revealed that the single-trial groups and the two massed trial groups are not different from one another but are both significantly different from the two spaced trial groups forry, Vol¹, andVol¹/+. The single-trial groups were different from the two-trial massed and two-trial spaced groups for Vol² andVol³/Vol².

Fig. 10.

Odor avoidance after mock conditioning. Odor avoidance to benzaldehyde or octanol was measured after mock training of ry and rut²⁰⁸⁰. Statistics are as follows. Two-factor ANOVA revealed no effect of genotype (F_(1,56) = 0.49; NS) or training procedure (F_(3,56) = 1.05; NS) using benzaldehyde as the odor. Two-factor ANOVA showed no effect of genotype (F_(1,56) = 0.94; NS) or training procedure (F_(3,56) = 0.28; NS) using octanol as the odor; n = 8 for all groups.