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The subjects were 77 male Wistar rats aged 8 weeks, acquired from Kyudo Co., Ltd. (Kumamoto, Japan). They were given free access to food and water, and housed two per cage for one week before the start of the experiments. Housing conditions were thermostatically controlled at 22–24°C with a light/dark cycle (lights on: 08:00—20:00). The experiments were performed under the control of the Ethics Committee of Animal Care and Experimentation in accordance with the Guiding Principles for Animal Care Experimentation, Kyushu Institute of Technology, Japan, and with the Japanese Law for Animal Welfare and Care.
The experiments took place in a test chamber consisting of two compartments, a light compartment (D25 cm×W25 cm×H27 cm) and a dark compartment (D30 cm×W30 cm×H30 cm) (Figure 1A). The two compartments were divided by a sliding door. Electric shocks are delivered by a shock generator (SGS-002, Muromachi Kikai Co., Ltd., Tokyo, Japan). In Experiment 2, a removable partition was used to prevent subject animals from moving from one compartment to the next earlier than the partners.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0006794.g001 The experimental design. (A) Experimental apparatus. (B) Time schedule of this study. The black arrow shows electric shock to the experienced subjects and partners (ES: experienced subjects; IS: inexperienced subjects; EP: experienced partners; IS: inexperienced partners). The gray arrow shows electric shock to the partners (SP: shocked partners). (C–E) Overview of the experiments. (C) The schematic diagram of the training session. The left row indicates the treatment for the subjects (ES: experienced subjects; IS: inexperienced subjects); the right row indicates the treatment for the partners (EP: experienced partners; IP: inexperienced partners). (D) The schematic diagram of Experiment 1. The upper row indicates interactive conditions, and the lower row indicates non-interactive conditions. (E) The schematic diagram of Experiment 2. The upper row shows non-interactive conditions, and the lower row shows interactive conditions.
All treatments or behavioral tests were done during the light cycle (12:00–20:00) in the following sequence (the whole schedule is shown in Figure 1B): 1. On the first day of this session (day 1), all animals were placed in the light compartment for 1 min individually and habituated to the experimental apparatus. After this interval, the sliding door was raised and the latency to enter the dark compartment was recorded. On the second day (day 2), a single electrical shock (0.5 mA, 5 s) was induced inescapably on 40 animals in the dark room after each animal entered the dark compartment, and they were used as the experienced subjects and 5 partners. The other 37 animals who received no electrical shocks were used as inexperienced subjects and partners. The experimental apparatus was cleaned with alcohol to remove odors before treating the next subject. On the third day (day 3), the latency of each animal to enter the dark compartment was measured. The schematic diagram of the training is shown in Figure 1C.
2. The subjects were divided into three groups: i) together with an experienced partner (EP), ii) with an inexperienced partner (IP), iii) without any partner (No). On the day following the training session (day 4), each subject was placed in the light compartment. If partnered, they were paired with the partner rats for 1 min. After the interval, the sliding door was raised and then the latencies to enter the dark compartment were measured, with a cut-off time of 15 min. This experiment was performed without any electric shocks. The schematic diagram of Experiment 1 is shown in Figure 1D.
3. The day after Experiment 1 was performed (day 5), experienced and inexperienced subjects were put in the experimental apparatus individually and habituated to the dark compartment for 20 min. On the second day of this experiment (day 6), 30 min before the test trial, each animal was placed in the light compartment and then the latencies to enter the dark compartment were measured, with a cut-off time of 5 min. We used the experienced subjects that entered within a given cut-off time as the experienced subjects (n = 16) and randomly selected inexperienced subjects (n = 12). In a test trial, each subject was placed in the light compartment with a partner for 1 min. Then, the sliding door was raised to permit the partners only to enter the dark compartment. During this time, a mesh partition attached in the center of the light compartment (in between a subject and a partner) did not permit the subjects to enter the dark compartment. After the partner entered, electrical shocks (0.5 mA, 3–6 s) were induced. Immediately after that, the partner returned to the light compartment and stayed there. After an additional interval (30 s), the partition was removed. The latencies to enter the dark compartment were measured with a cut-off time (15 min). The partner rat stayed in the light compartment and could interact with the subject freely during the measurement. We then compared the latency between the two conditions, with no partner and with a shocked partner. The schematic diagram of Experiment 2 is shown in Figure 1E. All partners were the rats already used in Experiment 1, which had been given a single foot shock to stabilize partners' pain reaction (habituation to the shock).
Data were analyzed with the use of SPSS software (version 16.0). Before analysis, the Kolmogorov-Smirnow test was performed for normality. In Experiment 1, we used the Turkey-Kramer multiple comparison test to assess the statistical significance of the difference among the rat groups. In Experiment 2, we used a paired t-test to evaluate the statistical significance of the difference between measurements in the absence and presence of partners. The criterion for statistical significance was p<0.05 (two-tailed).
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