The Zebrafish Dorsolateral Habenula Is Required for Updating Learned Behaviors

Abstract

Operant learning requires multiple cognitive processes, such as learning, prediction of potential outcomes, and decision-making. It is less clear how interactions of these processes lead to the behavioral adaptations that allow animals to cope with a changing environment. We show that juvenile zebrafish can perform conditioned place avoidance learning, with improving performance across development. Ablation of the dorsolateral habenula (dlHb), a brain region involved in associative learning and prediction of outcomes, leads to an unexpected improvement in performance and delayed memory extinction. Interestingly, the control animals exhibit rapid adaptation to a changing learning rule, whereas dlHb-ablated animals fail to adapt. Altogether, our results show that the dlHb plays a central role in switching animals' strategies while integrating new evidence with prior experience.

Keywords: behavioral flexibility; cognition; conditioned place avoidance; habenula; learning; memory consolidation; memory extinction; operant conditioning; reversal learning; zebrafish.

Graphical abstract

Figure 1

Juvenile Zebrafish Can Perform Conditioned Place Avoidance Learning with Increased Performance across Development (A) The top row shows a schematic representation of the protocol. B, baseline; C1, conditioning 1; T1, test 1; C2, conditioning 2; T2, test 2. Consecutive rows show the heatmaps depicting the average density of zebrafish position for each experimental group: 1-week-old (n = 36), 2-week-old (n = 24), 3-week-old (n = 24), and 4-week-old (n = 18) zebrafish. (B) Relative time spent in the conditioned zone (CZ). (C) Relative distance of swimming in the CZ. (D) Average distance of the animals away from the CZ. Dashed line indicates the midline. (E) Average swim velocity. (B–E) Line color and style indicate different age groups. Data displayed in 2-min bins; mean ± SEM; #, indicate statistical comparisons detailed in Figures S1E and S1F. (F) Histograms of CPA learning indices during the two test sessions. The darker colors represent the animals classified as learners. The percentage of learners is in the pie chart. See also Figures S1 and S2.

Figure 2

Comparing Operant Learning with Defensive Behaviors Elicited by Aversive Stimuli (A) The top row shows a schematic representation of the protocol. Consecutive rows show the heatmaps depicting the average density of zebrafish position for trained (n = 25) and sham (n = 25) groups. (B) Relative time spent in the CZ. (C) Relative distance of swimming in the CZ. (D) Average distance of the animals away from the CZ. Dashed line indicates the midline. (E) Average swimming velocity of the zebrafish. (B–E) Line colors indicate control (black) and sham (red) groups, 3- to 4-weeks old. Data are displayed in 2-min bins; mean ± SEM. (F) Percentage of time that the zebrafish exhibit no swimming less than 2-mm during 2 s. Solid lines represent median, and shaded areas represent first and third quartiles. (G) Average swimming velocity of the zebrafish, 1 sec before (b) and after (a) the delivery of aversive unconditioned stimuli (US). (H) Zebrafish swimming direction near the boundary of CZ and safe zone (SZ), during the last 5 min of C2 (left) and first 5 min of T2 (right), in CPA-trained (top, black) and sham (bottom, red) groups. Each dot on the polar plot depicts the average swimming direction of one zebrafish during the 5 secs after encountering the CZ/SZ boundary. For each dot, the distance from the center encodes the average swimming velocity of the animal. Filled dots represent successful avoidance of zebrafish, and empty dots represent unsuccessful ones. Dots outside the polar plots represent animals that never entered the CZ (filled) or that never left the CZ (empty). (I) Average ratio of successful avoidance over the protocol, divided in 5-min time bins (mean ± SEM). (J) Average swim duration up on each entry to CZ; data displayed in 5-min time bins; mean ± SEM. ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, ^∗p ≤ 0.05. Wilcoxon signed-rank test. See also Figure S3.

Figure 3

CPA Performance and Memory Recall Improve across Multi-day Training (A) The top row shows a schematic representation of the protocol. Blank, baseline with no color cues; R, recall. The next rows show the heatmaps depicting the average density of zebrafish position during multiple training days (n = 18). (B) Relative time spent in the CZ. (C) Relative distance of swim in the CZ. (D) Average distance of the animals away from the CZ. Dashed line indicates the animal being on the midline. (E) Average swimming velocity of the zebrafish. (B–E) Line colors and style indicate zebrafish performance during multiple training days in 4-week-old zebrafish. Data are displayed in 2-min bins; mean ± SEM; #, indicates significance comparisons, detailed in Figure S4A. (F) Histograms of the learning indices during the test session, across multiple days of training. The darker colors represent the animals classified as learners. The percentage of learners is in the pie chart. (G) Average time spent by the animal in the CZ during the last 10 min of the blank session (B’) and in the first 10 min of the recall session (R’). (H) Relative distance of swim in the during B’ and in R’. B’ and R’ periods are marked with the gray box in (B) and (C). (I) Average ratio of successful avoidance over the course of the protocol, divided in 5-min time bins; mean ± SEM. ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, ^∗p ≤ 0.05. (G, H, and K) Wilcoxon signed-rank test. See also Figure S4.

Figure 4

Dorsolateral Habenula Ablation Improves CPA Performance and Delays Memory Extinction (A and B) Confocal microscopy images of Tg(narp:Gal4;UAS-E1b:NTR-mCherry) zebrafsh before (A) and after (B) metrodiniazole (MTZ) treatment. Dorsal (top) and coronal (bottom) views. Scale bars are 25 μm. (C, F, I, and L) The top row shows a schematic representation of the protocol. The next rows show the heatmaps depicting the average density of zebrafish position for dlHb-ablated (n = 11) and control (n = 12) groups. (C) First day of training. (F) Second day of training. (I) Third day of training. (L) Fourth day of training. (D, G, J, and M) Relative time spent in the CZ. (E, H, K, and N) Average distance of the animals from the CZ. Dashed line indicates the midline. (D, E, G, H, J, K, M, and N) Line colors indicate control (black) and dlHb-ablated (red) groups of 3- to 4-week-old zebrafish. Data are displayed in 2-min bins; mean ± SEM. (O) Relative time spent by the dlHb-ablated zebrafish in the CZ during the last 10 min of the B’ and in the first 10 min of the R’. (P) Relative distance swam in the CZ during B’ and R’. ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, ^∗p ≤ 0.05. (D, E, G, H, J, K, M, and N) Wilcoxon rank-sum test; (O and P) Wilcoxon signed-rank test. See also Figure S5.

Figure 5

The Dorsolateral Habenula Is Important for Behavioral Flexibility during Reversal Learning (A) The top row shows a schematic representation of the protocol. T3, test 3 (extinction test); C3, conditioning 3 (reversal learning); T4, test 4 (reversal learning test); C4, conditioning 4 (reversal learning); T5, test 5(reversal learning test); T6, test 6(reversal learning extinction test). The next rows show the heatmaps depicting the average density of zebrafish position for control (n = 21) and dlHb-ablated (n = 22) groups. (B) Relative time spent in the CZ. (C) Average distance of the animals away from the CZ. Dashed line indicates the midline. (D) The ratio of swimming distance in CZ versus SZ. (E) Average swimming velocity of the zebrafish. (B–E) Line colors indicate control (black) and dlHb-ablated (red) groups of 3- to 4-week-old zebrafish. Data are displayed in 2-min bins; mean ± SEM. ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, ^∗p ≤ 0.05, Wilcoxon rank-sum test. See also Figure S5.