Associative learning in zebrafish (Danio rerio) in the plus maze

Abstract

Zebrafish has been gaining increasing amount of interest in behavioral neuroscience as this species may represent a good compromise between system complexity and practical simplicity. Particularly successful have been those studies that utilized zebrafish as a screening tool. Given the complexity of the mechanisms of learning, for example, forward genetic screens with zebrafish could potentially reveal previously unknown genes and molecular pathways that subserve this function. These screens, however, require appropriate phenotypical (e.g. behavioral) paradigms. A step in this direction is the characterization of learning abilities of zebrafish. Here we employ two classical learning tasks in a plus maze. In the first, zebrafish are required to associate a visible cue with food reward irrespective of the location of this pairing. In the second, zebrafish are required to associate the spatial location of food reward irrespective of intra-maze cues. Our results demonstrate that zebrafish perform well in both tasks and show significant acquisition of the association between cue and reward as well as between location and reward. We conclude that zebrafish, similar to classical laboratory rodents, may have utility in the biological analysis of simple as well as complex forms of associative learning.

Figure 1

The plus maze (4 arm radial maze). Note that each arm is equipped with a food dispensing syringe (grey rectangles) attached to Teflon tubing. The end of the Teflon tube may be positioned just behind or just in front of perforated plastic sheets (broken line). If the end of the tube is in front of the perforated sheet, the fish have access to food. Also note the center square piece (thick black line), which allowed us to place the fish in the maze and which could be lifted remotely using a pulley and nylon string system thereby allowing the fish to start the exploration of the maze with minimal experimenter interference.

Figure 2

Simple associative learning: Percent of time in the target arm significantly differs between the groups at the probe trial. Panel A, performance at the first training trial. Panel B, performance at the probe trial, i.e. after 20 training trials. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 10. The solid horizontal line represents random chance level performance. Significant difference (p < 0.01) between the groups is indicated by two asterisks. For methodological details see Methods. For details of statistical analyses, see Results.

Figure 3

Simple associative learning: Frequency of target arm visits relative to total number of arm visits, expressed as percentage, significantly differs between the groups at the probe trial. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 10. The single asterisk indicates significant difference at p < 0.05. For methodological details see Methods. For details of statistical analyses, see Results.

Figure 4

Simple associative learning: The total number of arm visits significantly differs between the groups at the probe trial. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 10. The single asterisk indicates significant difference at p < 0.05. For methodological details see Methods. For details of statistical analyses, see Results.

Figure 5

Spatial learning: Percent of time in the target arm significantly differs between the groups at the probe trial. Panel A, performance at the first training trial. Panel B, performance at the probe trial, i.e. after 20 training trials. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 12. The solid horizontal line represents random chance level performance. Significant difference (p < 0.05) between the groups is indicated by an asterisk. For methodological details see Methods. For details of statistical analyses, see Results.

Figure 6

Spatial learning: Frequency of target arm visits relative to total number of arm visits, expressed as percentage, does not significantly differ between the groups at the probe trial. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 12. For methodological details see Methods. For details of statistical analyses, see Results.

Figure 7

Spatial learning: The total number of arm visits does not significantly differ between the groups at the probe trial. Mean ± SEM are shown. Sample sizes n_unpaired = 10, n_paired = 12. For methodological details see Methods. For details of statistical analyses, see Results.