Effect of simultaneous testing of two mice in the tail suspension test and forced swim test

Abstract

In mouse studies, the results of behavioural experiments are greatly affected by differences in the experimental environment and handling methods. The Porsolt forced swim test and tail suspension test are widely used to evaluate predictive models of depression-like behaviour in mice. It has not been clarified how the results of these tests are affected by testing single or multiple mice simultaneously. Therefore, this study evaluated the differences between testing two mice simultaneously or separately. To investigate the effect of testing multiple mice simultaneously, the Porsolt forced swim test and tail suspension test were performed in three patterns: (1) testing with an opaque partition between two mice, (2) testing without a partition between two mice, and (3) testing a single mouse. In the Porsolt forced swim test, the mice tested simultaneously without a partition demonstrated increased immobility time as compared to mice tested alone. No difference in immobility time was observed between the three groups in the tail suspension test. Our results showed that the environment of behavioural experiments investigating depression-like behaviour in mice can cause a difference in depression-like behaviour. The results of this experiment indicated that it is necessary to describe the method used for behavioural testing in detail.

Figure 1

Schematic illustration of the experimental apparatus. (a) Tail suspension test. (b) Porsolt forced swim test.

Figure 2

Simultaneous tail suspension testing of two mice. (a) Schematic diagram of the experiment. Graphs showing the total number of immobility episodes (b) and the immobility episodes during each 1-min period (c). Graphs showing the total time spent immobile (d) and the proportion of time spent immobile during each 1-min period (e). All data are presented as box plots (b, d) or means ± standard errors (c, e). Statistical significance is represented by asterisks: *p < 0.05, ⁺p < 0.1. The p values were calculated using one-way analysis of variance (analysis of variance [ANOVA] in (b) and (d) and two-way repeated-measures ANOVA in (c) and (e). SEM: standard error of the mean. individual: n = 10, opaque partition: n = 10, no partition: n = 10. Respective mean and SEM are listed in Table 1.

Figure 3

Simultaneous Porsolt forced swim test of two mice on day 1. (a) Schematic diagram of the experiment. Graphs showing the total number of immobility episodes (b) and the immobility episodes during each 1-min period (c). Graphs showing the total time spent immobile (d) and the proportion of time spent immobile during each 1-min period (e). All data are presented as box plots (b, d) or means ± standard errors (c, e). Statistical significance is represented by asterisks: *p < 0.05, ⁺p < 0.1. The p values were calculated using one-way analysis of variance (ANOVA) in (b) and (d) and two-way repeated-measures ANOVA in (c) and (e). SEM: standard error of the mean. individual: n = 10, opaque partition: n = 10, no partition: n = 10. Respective mean and SEM are listed in Table 2.

Figure 4

Simultaneous Porsolt forced swim test of two mice on day 2. Graphs showing the total number of immobility episodes (a) and the immobility episodes during each 1-min period (b). Graphs showing the total time spent immobile (c) and the proportion of time spent immobile during each 1-min period (d). All data are presented as box plots (a, c) or means ± standard errors (b, d). Statistical significance is represented by asterisks: *p < 0.05, ⁺p < 0.1. The p values were calculated using one-way analysis of variance (ANOVA) in (a) and (c) and two-way repeated-measures ANOVA in (b) and (d). SEM: standard error of the mean. individual: n = 10, opaque partition: n = 10, no partition: n = 10. Respective mean and SEM are listed in Table 3.

Figure 5

Behaviour due to the nature of the demonstrator inside the cylinder in the Porsolt forced swim test. (a) Schematic diagram of the experiment. Graphs showing the total number of immobility episodes (b) and the immobility episodes during each 1-min period (c). Graphs showing the total time spent immobile (d) and the proportion of time spent immobile during each 1-min period (e). All data are presented as box plots (b, d) or means ± standard errors (c, e). Statistical significance is represented by asterisks: *p < 0.05, ⁺p < 0.1. The p values were calculated using one-way analysis of variance (ANOVA) in (b) and (d) and two-way repeated-measures ANOVA in (c) and (e). SEM: standard error of the mean. control: n = 10, high immobility: n = 10, imipramine: n = 10, stationary black toy: n = 10, moving black toy: n = 10. Respective mean and SEM are listed in Table 4.