Anti-instinctive Learning Behavior Revealed by Locomotion-Triggered Mild Heat Stress in Drosophila

Abstract

Anti-instinctive learning, an ability to modify an animal's innate behaviors in ways that go against one's innate tendency, can confer great evolutionary advantages to animals and enable them to better adapt to the changing environment. Yet, our understanding of anti-instinctive learning and its underlying mechanisms is still limited. In this work, we describe a new anti-instinctive learning behavior of fruit flies. This learning paradigm requires the fruit fly to respond to a recurring, aversive, mild heat stress by modifying its innate locomotion behavior. We found that experiencing movement-triggered mild heat stress repeatedly significantly reduced walking activity in wild type fruit flies, indicating that fruit flies are capable of anti-instinctive learning. We also report that such learning ability is reduced in dopamine 1-like receptor 1 (Dop1R1) null mutant and dopamine 2-like receptor (Dop2R) null mutant flies, suggesting that these two dopamine receptors are involved in mediating anti-instinctive learning in flies.

Keywords: Drosophila; dopamine receptors; learning; operant conditioning; stress.

Figure 1

Behavioral experiment design. (A) A schematics of a LaserBox design along with an infrared laser emitter and a condenser. The large box shown on the left is the LaserBox, inside which are (from bottom to top) a red LED array, a diffuser, a glass tube with a fly in it, and a short-pass filter-covered position sensor. The red arrows illustrate light paths of the red lights from the LED array. The gray arrows represent light paths of the infrared light from the laser emitter. The LaserBox circuit board is not shown. (B) A photo of the actual LaserBox. The layout of the setup is identical to the schematics shown in panel A, except that the diffuser is positioned closer to the LaserBox than depicted in (A), and that the position sensor is installed inside the black fixture, rendering it invisible in this picture. The green circuit board is the LaserBox circuit board not shown in (A). (C) A 40-min recording of a fly's spontaneous activity in a LaserBox. The fly's spatial trajectory inside the tube is illustrated in the right diagram. (D) An example of a fly's body temperature profile when the fly is being irradiated by the laser emitter. The dotted line indicates when the laser emitter is turned on. The measurement of the fly's body temperature can be found in the Materials and Methods section. (E) Behavioral paradigm used in this study. The paradigm consists of 5 sessions: Pre-test, Train 1, Test 1, Train 2, and Test 2 sessions. The flies in the experimental group is called Train Flies, and flies in the two control groups are called Yoked control flies and Blank control flies. The timing relationship between a fly's walking activity and the status of the laser emitter in that fly's chamber is illustrated in the diagram, in which laser ON is labeled with red, and walking activity is labeled with gray.

Figure 2

The train flies reduce activity during the train sessions. Sample size: 64 (Train flies), 125 (Yoked control flies), 72 (Blank control flies). (A) Likelihood of receiving heat stress when a fly is at different behavioral states during Train 1 session. The bright red box corresponds to the likelihood of Train flies receiving heat stress during walking. The light red box corresponds to the likelihood of Train flies receiving heat stress during pause. The bright blue box represents the likelihood of Yoked control flies receiving heat stress during walking. The light blue box represents the likelihood of Yoked control flies receiving heat stress during pause. (B) Flies' cumulative active duration (CAD) during Train 1 session. Data from the Train flies is shown in red, while the Yoked control fly data and the Blank control fly data are shown in blue and gray, respectively. The solid lines represent the median in each group, and the shaded regions correspond to the confidence intervals. The CAD of Train flies at the end of Train 1 session (the 163th sec) is significantly lower than the CAD of other two groups (p < 0.05, Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests) (C) Likelihood of receiving heat stress when a fly is at different behavioral states during Train 2 session. The color reference is identical to that of (A). (D) CAD during Train 2 session. The color reference is identical to that of (B). The CAD of Train flies at the end of Train 2 session (the 163th sec) is significantly lower than the CAD of other two groups (p < 0.0001, Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests). ****p < 0.0001.

Figure 3

Train flies' continue to show less activity after training ends. Sample size: 64 (Train flies), 125 (Yoked control flies), 72 (Blank control flies). (A) Train, Yoked control, and Blank control flies' activity levels (percentage of time a fly is active during the entire test session) in Pre-Test, Test 1 and Test 2 sessions. During Pre-Test session, all three groups of flies show comparable activity levels during Pre-Test. During each subsequent test session (Test 1 and Test 2), Train flies and Yoked control flies activity levels decreased significantly (Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests). (B) All three groups of flies' activity difference (AD, the change of activity level shown in A) of Test 1 and Test 2 from that of Pre-Test (i.e., the AD of Test 1 is the activity level of Test 1 minus the activity level of Test 2. To avoid confusion, here we do not use the percentage as the unit). The AD showed that the activity level of Train flies decreases significantly more than that of the two control groups (Kruskal-Wallis test followed by pairwise Wilcoxon rank sum tests). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

Yoked control flies show a moderate decrease in activity level. Sample size: 125. Correlation coefficient, slope, and the statistical significance are shown on the top right corner. (A) Correlation between AD and total laser exposure. (B) Correlation between AD and ED after Train 1 session. (C) Correlation between AD and ED after All Train sessions. **p < 0.01.

Figure 5

Anti-instinctive learning performance in dopamine receptor null mutants. (A–D) The expression patterns of the dopamine receptors (anterior view, scale bar: 100 μm). (A) Dop1R1. (B) Dop1R2. (C) DopEcR. (D) Dop2R. (E) Anti-instinctive learning performance in dopamine receptor null mutants. Dop1R1 and Dop2R are involved in flies' anti-instinctive learning revealed by the activity difference (AD) between Pre-Test and Test 2. Sample sizes are indicated at the top of the graph. *p < 0.05.