Context and occasion setting in Drosophila visual learning

Abstract

In a permanently changing environment, it is by no means an easy task to distinguish potentially important events from negligible ones. Yet, to survive, every animal has to continuously face that challenge. How does the brain accomplish this feat? Building on previous work in Drosophila melanogaster visual learning, we have developed an experimental methodology in which combinations of visual stimuli (colors and patterns) can be arranged such that the same stimuli can either be directly predictive, indirectly predictive, or nonpredictive of punishment. Varying this relationship, we found that wild-type flies can establish different memory templates for the same contextual color cues. The colors can either leave no trace in the pattern memory template, leading to context-independent pattern memory (context generalization), or be learned as a higher-order cue indicating the nature of the pattern-heat contingency leading to context-dependent memory (occasion setting) or serve as a conditioned stimulus predicting the punishment directly (simple conditioning). In transgenic flies with compromised mushroom-body function, the sensitivity to these subtle variations is altered. Our methodology constitutes a new concept for designing learning experiments. Our findings suggest that the insect mushroom bodies stabilize visual memories against context changes and are not required for cognition-like higher-order learning.

Figure 1.

Flight simulator set-up and experimental schematics. (Left) The fly is flying stationarily in a cylindrical arena homogeneously illuminated from behind. The fly’s tendency to perform left or right turns (yaw torque) is measured continuously and fed into the computer. The computer controls arena position, IR-laser (heat) activation and color of illumination according to the conditioning rules. (Right) Experimental schematics used in this study. Patterns and colors depict the wall of the cylinder surrounding the fly. Colored boxes indicate the four 90° quadrants. Red boxes in the example color/pattern schematics depict heated quadrants. Note that even though quadrants may be drawn in different colors here, the illumination of the entire arena is always changed. See Materials and Methods for a detailed description.

Figure 2.

Colors can be context, occasion setters, and conditioned stimuli, depending on the temporal arrangement with the unconditioned stimulus. (A,B,C) Color as context. The color of background illumination during operant visual pattern discrimination learning changes according to the experimental schedule. (A) Context generalization. A single change of background illumination after the final training period marks the beginning of a 2-min test period for pattern memory in the new background color. The flies are able to show pattern memory in the new context. (B) The color changes are concomitant with the change from training to test periods. Increasing the number of context changes with respect to A abolishes the generalization effect —feature discrimination. (C) Control group in which training and test periods alternate in constant background color. Alternating training and test periods as in B does not abolish the memory score. (D–G) Color as occasion setter. (D) Color changes indicate the reversal of the pattern-heat contingency—temporal conditional discrimination. Reversal learning cannot be facilitated by context changes. (E,F,G) Colors change independently of the experimental schedule and indicate heated quadrants in conjunction with visual patterns. (E) Flies can solve a fully operant spatial conditional discrimination paradigm. (F) Flies fail to solve a temporal conditional discrimination task, where the color presentations are yoked to the animals in E. (G) Flies can solve spatial conditional discrimination with classical training and operant test periods. (H) Color as CS. The colors used in this study can be discriminated by wild-type flies in an operant visual learning task, with the colors as conditioned stimuli. Colored boxes with patterns illustrate the experimental design as in Figure 1 (see Materials and Methods for details). White/gray squares indicate 1-min. periods, rectangles 2-min.periods in the experimental time course. The performance indices of the highlighted test periods (bold) are displayed in the bar-graphs on the right. ***P < 0.001; **P < 0.01; *P < 0.05; (n.s.) not significant. Numbers next to bar graphs indicate number of animals. Lines under experimental periods (indicated by “TR” in A) denote training periods. Performance index: PI = (t_a-t_b)/(t_a+t_b).

Figure 3.

Operant conditional discrimination learning is not due to simple conditioning. (A) Summed fixation histograms of the last 7 min for all 26 flies in the operant conditional discrimination experiment depicted in Figure 2E. (Left) Relative time spent at flight directions of 0–360° with respect to one of the hot/cold borders during the last 5 min of training. (Right) Flight directions during the following two minutes of test. The flies fixate the patterns preferentially both during training and during test. (Shaded area) Quadrants associated with heat; (white area) quadrants associated with heat-off; (dashed lines) quadrant borders between T-patterns. (B) Performance index of a control experiment in which patterns and colors were arranged as in an operant spatial conditional discrimination experiment (Fig. 2E), but with the T-shaped patterns replaced by four identical stripes as in color discrimination learning (Fig. 2H). No significant performance index was obtained. Performance index: PI = (t_a-t_b)/(t_a+t_b).

Figure 4.

Flies with blocked mushroom-body output fail in context generalization and temporal conditional discrimination, but perform in spatial conditional discrimination. (A) Context generalization as in Figure 2A. Flies with blocked mushroom-body output do not transfer the pattern memory acquired during training to a different background color. (B) Temporal conditional discrimination where the colors in each period indicate the nature of the pattern/heat contingency (as in Fig. 2D). As in wild-type flies, color changes do not facilitate reversal learning and, hence, are not learned as occasion setters in flies with blocked mushroom-body output. (C) Operant spatial conditional discrimination as in Figure 2E, but with altered period set-up and duration. Colored boxes with patterns illustrate the experimental design as in Figure 1 (see Materials and Methods for details). White/gray rectangles indicate 2-min. periods, as in Figure 2. Performance indices of highlighted (bold) test periods are displayed in the bar-graphs on the right. **P < 0.01; (n.s.) not significant. Numbers next to bar graphs indicate number of animals. Lines under experimental periods (indicated by “TR” in A) denote training periods. Performance index: PI = (t_a-t_b)/(t_a+t_b).