Animals use salient cues to navigate in their environment, but their specific cognitive strategies are largely unknown. We developed a conditioned place avoidance paradigm to discover whether and how zebrafish form spatial memories. In less than an hour, juvenile zebrafish, as young as 3 weeks, learned to avoid the arm of a Y-maze that was cued with a mild electric shock. Interestingly, individual fish solved this task in different ways: by staying in the safe center of the maze or by preference for one, or both, of the safe arms. In experiments in which the learned patterns were swapped, rotated, or replaced, the animals could transfer the association of safety to a different arm or to a different pattern using either visual cues or location as the conditioned stimulus. These findings show that juvenile zebrafish exhibit several complementary spatial learning modes, which generate a flexible repertoire of behavioral strategies.

15Animals use salient cues to navigate in their environment, but their specific cognitive 16 strategies are largely unknown. We developed a conditioned place avoidance paradigm 17 to discover whether and how zebrafish form spatial memories in a Y-shaped maze. 18 Juvenile zebrafish, older than three weeks, learned to avoid the arm of the maze that 19 was cued with a mild electric shock. We found that the fish required distinct visual 20 patterns to develop a conditioned response. Interestingly, individual fish solve this task 21 in different ways: by staying in the safe center of the maze, by preference for one, or 22 both, of the safe patterns, or by mixed strategies. In experiments in which the learned 23 patterns were swapped, rotated or replaced, the animals could transfer the association 24 of safety to a different arm or to a different pattern using either visual cues or location 25 as the conditioned stimulus. These findings show that juvenile zebrafish exhibit several 26 complementary spatial learning modes and pave the way for neurobiological studies of 27 navigational mechanisms in this model species. 28 29 104 105A biologically realistic agent model replicates learned avoidance behavior 106 Larval and juvenile zebrafish swim in characteristic swim bouts (Supplementary Video 1). 107Electric stimulation caused swim bouts of increased amplitude compared to the amplitude of 108 spontaneous swim bouts ( Figure 1E). We hypothesized that increased swimming speeds 109 under electric stimulation in the conditioned arm could lead to changes in the OC and EF 110 measures independent of the learning abilities of the fish. We developed a null-model of the 111 6 112 Figure 1. The conditioned place avoidance (CPA) paradigm.113 (A) Experimental setup. (B) Measures of fish performance in the paradigm, top: arm occupancy (OC); bottom: 114 arm entry frequency (EF); middle: schematic for the calculation of the preference scores for OC/EF. Positive 115 values of the scores correspond to arm preference, negative values correspond to arm avoidance. Red color 116 represents the shocked arm, black and gray represent the safe arms. (C) Left: moving averages of the OC/EF 117 scores in a 2-hour control experiment (mean ± s.e.m.). Right: box plots for OC/EF scores in the first and the last 118 5 minutes of the control experiments (Mann-Whitney test, n = 24 fish). Box plots show median and quartiles; 119 whiskers show 1.5x interquartile range; dots show values for individual fish. (D) There is no significant 120 preference for any of the visual patterns (ANOVA, n = 24 fish). (E) Distributions of amplitudes for spontaneous 121 (gray) and shock-triggered swim bouts (red). (F) Pseudo-random 1D walk model used to evaluate CPA 122 measures. Bout amplitude S for simulated movement was drawn from a Gamma distribution, amplitude in the 123 conditioned arm was multiplied by a speed ratio α. (G) Top: model without learning. Bottom: model with 124 learning. Learning rule was implemented by decreasing the probability of entry into t...