Single stimulus learning in zebrafish larvae

Abstract

Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7dpf larvae and extended the finding to 5 and 6dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed.

Keywords: Activity; Avoidance; Habituation; Learning; Thigmotaxis; Zebrafish larvae; t1–t2 Design.

Figure 1. The zebrafish imaging system

A) Camera mounted on top shelf of cabinet with laptop on bottom. B) Camera is mounted on an L-shaped bracket attached to top shelf. C) A laptop is used to display PowerPoint presentations to the larvae in multiwell plates. D-G) PowerPoint slides used for the single stimulus learning assay. D and E are the input (t1) slides that alternated every 5 min. D also served as the set up template with white disks to ensure that the multiwell plates were situated correctly. E is the stimulus exposure slide with gray bouncing disks on one side and gray stationary disks on the other (sides are counterbalanced across larvae). F and G are the two 5 min test (t2) slides. F is identical to D and immediately preceded G. In G, the gray bouncing disk appeared in all wells. In this example, the larvae on the left had been exposed to the moving disk but those on the right had not. H-K) Image analysis. Images were first opened in ImageJ (H), a region of interest (yellow ring) was set to encompass the entire swimming area, and larvae were separated from the larger disk using ImageJ’s particle analysis (I). When a larva overlapped with the disk (J), background subtraction was applied and a region of interest was placed around the larva (K). Scale bar in K = 1 cm.

Figure 2. Experiment 1

Mean responses in testing (t2) to the gray bouncing disk for 7 dpf larvae that had been exposed in training (t1) either to that gray bouncing disk (Group B, black bars) or to an identical but stationary disk (Group S, gray bars). Data are shown separately for the 5-min period with no visual stimulus (White) and the 5-min period with the gray bouncing disk (GBD). A) Mean % avoidance denotes the percent observations that the larva was on the side away from where the stimulus was presented; B) Mean % still denotes percent displacements of larval centroids between two consecutive images that were smaller than 0.3 mm.

Figure 3. Experiment 2: Effects of larval age

Mean responses in testing (t2) to the gray bouncing disk for larvae that had been exposed in training (t1) either to that gray bouncing disk (Group B, black bars) or to an identical but stationary disk (Group S, gray bars). Data are shown separately for the 5-min period with no visual stimulus (White) and the 5-min period with the gray bouncing disk (GBD). A, C, E) Mean % avoidance denotes the percent observations that the larva was on the side away from where the stimulus was presented for 7, 6, and 5 dpf larvae, respectively; B, D, F) Mean % still denotes percent displacements of larval centroids between two consecutive images that were smaller than 0.3 mm for 7, 6, and 5 dpf larvae, respectively.

Figure 4. Experiment 3: Effects of stimulus exposure

Mean responses in testing (t2) to the gray bouncing disk for larvae that had been exposed in training (t1) either to that gray bouncing disk (Group B, black bars) or to an identical but stationary disk (Group S, gray bars). Data are shown separately for the 5-min period with no visual stimulus (White) and the 5-min period with the gray bouncing disk (GBD). A, C, E) Mean % avoidance denotes the percent observations that the larva was on the side away from where the stimulus was presented following 8, 4, or 1 stimulus presentations, respectively; B, D, F) Mean % still denotes percent displacements of larval centroids between two consecutive images that were smaller than 0.3 mm following 8, 4, or 1 stimulus presentations, respectively.