Development of a Modified Three-Day T-maze Protocol for Evaluating Learning and Memory Capacity of Adult Zebrafish

Abstract

A T-maze test is an experimental approach that is used in congenital research. However, the food reward-based protocol for the T-maze test in fish has low efficiency and a long training period. The aim of this study is to facilitate the T-maze conditions by using a combination of the principles of passive avoidance and a spatial memory test. In our modified T-maze settings, electric shock punishment (1-2 V, 0.3-0.5 mA) is given at the left arm, with a green cue at the right arm. Also, the depth of both arms of the T-maze was increased. The parameters measured in our T-maze design were latency, freezing time, and time spent in different areas of the T-maze. We validated the utility of our modified T-maze protocol by showing the consistent finding of memory impairment in ZnCl₂-treated fish, which has been previously detected with the passive avoidance test. In addition, we also tested the spatial memory performance of leptin a (lepa) mutants which displayed an obesity phenotype. The results showed that although the learning and memory performance for lepa KO fish were similar to control fish, they displayed a higher freezing behavior during the training phase. In conclusion, we have established a modified T-maze protocol that can be used to evaluate the anxiety, learning, and memory capacity of adult zebrafish within three days, for the first time.

Keywords: T-maze; ZnCl2; leptin a; passive avoidance; spatial memory; zebrafish.

Figure 1

Experimental design of the T-maze apparatus in the zebrafish model. (A) Illustration of the T-maze dimensions. (B) Three sections were defined: the start chamber (Zone I), the novel arm (Zone II), and the choice arm (swim to Zones III and IV). The depth of water in the novel arm was maintained at 35 and 85 mm in two of the deeper choice arms (C) The T-maze was equipped with an electric shock device (1–2 V, 0.3–0.5 mA), video recorder, and the data were analyzed by GraphPad Prism. (D) Flow diagram of the T-maze protocol.

Figure 2

The assessment of individual fish with color cue option in the habituation phase. (A) Three T-maze designs. (B) The comparison of percentage fish entry to each arm in three different T-maze configurations. Based on the results, we decided to use the T-maze apparatus with a green cue at the right arm and electric shock at the left arm (punishment chamber). The data expressed as the means ± SEM. The data were analyzed by one-way ANOVA and followed with the Tukey–HSD test (* p < 0.05, **** p < 0.0001, n = 48).

Figure 3

The assessment of the influence of gender on learning and memory in the T-maze test. (A) Comparison of the latency (s) for male (blue) and female (red) fish to swim into the punishment chamber for each training session. (B) Comparison of the time spent in punish arm (s) for male and female fish at different time points before and after training. (C) Comparison of the freezing time (s) for male and female fish at different time points before and after training. (D) Comparison of the total number of electric shock between male and female fish. (E) Comparison of the memory retention latency (s) for male and female fish at different time points after training. The data are expressed as means ± SEM. The data were analyzed by two-way ANOVA and followed with the Tukey–HSD test. Different letters (a, b, c) on the error bars represent significant differences (p < 0.05, n = 18).

Figure 4

The assessment of toxicity of ZnCl₂ exposure on learning and memory performances in the T-maze test. (A) The latency (s) of control and ZnCl₂-incubated fish to swim into the punishment chamber for each training session. (B) The time spent in the punishment chamber (s) of control and ZnCl₂-incubated fish at different time points (before and after training). (C) The freezing time (s) of control and ZnCl₂-incubated fish at different time points before and after training. (D) The total number of electric shocks given for control and ZnCl₂-incubated fish. (E) The memory retention latency (s) of control and ZnCl₂-incubated fish at different time points after training. (F) The typical swim locomotion trajectories for control fish before (left panel) and after training (middle panel), and for ZnCl₂ exposed fish after training (right panel). The data are expressed as means ± SEM. The data were analyzed by two-way ANOVA and followed with the Tukey–HSD test (** p < 0.01, n = 18). Different letters (a, b, c) on the error bars represent significant differences (p < 0.05).

Figure 4

The assessment of toxicity of ZnCl₂ exposure on learning and memory performances in the T-maze test. (A) The latency (s) of control and ZnCl₂-incubated fish to swim into the punishment chamber for each training session. (B) The time spent in the punishment chamber (s) of control and ZnCl₂-incubated fish at different time points (before and after training). (C) The freezing time (s) of control and ZnCl₂-incubated fish at different time points before and after training. (D) The total number of electric shocks given for control and ZnCl₂-incubated fish. (E) The memory retention latency (s) of control and ZnCl₂-incubated fish at different time points after training. (F) The typical swim locomotion trajectories for control fish before (left panel) and after training (middle panel), and for ZnCl₂ exposed fish after training (right panel). The data are expressed as means ± SEM. The data were analyzed by two-way ANOVA and followed with the Tukey–HSD test (** p < 0.01, n = 18). Different letters (a, b, c) on the error bars represent significant differences (p < 0.05).

Figure 5

The comparison of learning and memory capabilities between WT and lepa KO fish. (A) Comparison of the latency (s) of control (red) and lepa KO (blue) fish to swim into the punishment chamber for each training session. (B) Comparison of the time spent in punish arm (s) for control and lepa KO fish at different time points before and after training. (C) Comparison of the freezing time (s) for control and lepa KO fish at different time points before and after training. (D) Comparison of the total number of electric shocks between control and lepa KO fish. (E) Comparison of the memory retention latency (s) for control and lepa KO fish at different time points after training. The data are expressed as mean ± SEM and bars with the same letter are not significantly different from each other. (A) the data were tested by one-way ANOVA with Tukey–HSD post hoc test; (B–E) the data were tested by two-way ANOVA with Tukey–HSD post hoc test (* p < 0.05, **** p < 0.0001, n = 18). Different letters (a, b, c) on the error bars represent significant differences (p < 0.05).