Ged 7 weeks and weighing 22.161.3 g, were used for this study. The

Ged 7 weeks and weighing 22.161.3 g, were used for this study. The mice were randomly divided into five groups: control mice (C mice; n = 10), tryptophandeficiency (TD) mice (TD mice; n = 10), TD+chronic inhibitor unpredictable stress (CUS) mice (TD+CUS mice; n = 10), TD+CUS+moderate exercise (ME) mice (TD+CUS+ME mice; n = 10) and TD+CUS+intense exercise (IE) mice (TD+CUS+IE mice; n = 10). As our previous study [16] indicated that mice fed a normal diet with exposure to CUS showed depressive behavior, in this study, we omitted the experimental condition concerning the mice fed a normal diet with exposure to CUS. The C and TD mice were housed in standard 15481974 mouse cages with five mice per cage. The TD+CUS, TD+CUS+ME and TD+CUS+IE mice were housed in standard mouse cages divided into six cells to reduce their living space and to decrease their daily activity [17]. The TD, TD+CUS, TD+CUS+ME and TD+CUS+IE mice were fed a tryptophandeficient (TD) powered diet (Oriental Yeast Co., Ltd., Japan). The C mice were fed a TD powered diet supplemented with tryptophan at 214 mg per 100 g of powder diet, equal to the tryptophan content in a standard animal diet. All diets were mixed with hot water, kneaded, cut and dried to make hard pellets. Tap water was given to all mice. All mice were introduced to their respective diets for a week before the start of the CUS procedure. During the experimental period, all mice were allowed to eat and drink ad libitum. The weight of all mice was measured once a week throughout the experimental period.5. Behavioral Tests(1) Forced swimming test (FST). All mice were subjected to FST to evaluate depression-like behavior according to the method of Porsolt et al. [19]. For testing, cylinders (height, 2 cm; diameter, 15 cm) filled with water (25uC) were used to make the mice swim or float without touching their hindlimbs or tail on the bottom of the cylinder. Each mouse was individually placed in the cylinder and its movements were recorded for 6 min using a video camera. Immobility time, when the mouse performed the minimal movement required to stay afloat, was measured to evaluate depression-like behavior during the latter four minutes of the test. (2) Sucrose preference test (SFT). At the end of 28 days of CUS, all mice were subjected to SFT to evaluate depression-like behavior according to the method of Sakata et al. [20]. In brief, after animals had been habituated to two water bottles for 3 days in their home cages, a free choice between plain water and 12. Chronic Unpredictable StressThe timeline of the various experimental Epigenetic Reader Domain procedures is shown in Fig. 1. After 7 days of acclimation to cage and diet, all mice wereFigure 1. Experimental procedures. Habituation: habituation to cage, food and treadmill running; CUS: chronic unpredictable stress; PAT: passive avoidance test; ORT: object recognition test; SFT: sucrose preference test; FST: forced swimming test. doi:10.1371/journal.pone.0066996.gExercise Prevents Depression in TD MiceTable 1. Protocol of chronic unpredictable stress.Day time stress Immobilization; 3 h Cold isolation(uC); 3 hTimes 6Overnight stress Light on overnigh Web bedding overnigh Crowding overnight Food/water deprivation overnight Tilt of cage Stroboscope overnight TotalTimes 5 4 4 4 5 5Cage rotation (100 rpm); 3 h 5 Swim in water(18uC); 5 min 3 Rat odor; 3 hConfrontation with rat; 3 h 5 Totaldoi:10.1371/journal.pone.0066996.tsucrose solution was provided to each mouse. The positions of the bottles were counterb.Ged 7 weeks and weighing 22.161.3 g, were used for this study. The mice were randomly divided into five groups: control mice (C mice; n = 10), tryptophandeficiency (TD) mice (TD mice; n = 10), TD+chronic unpredictable stress (CUS) mice (TD+CUS mice; n = 10), TD+CUS+moderate exercise (ME) mice (TD+CUS+ME mice; n = 10) and TD+CUS+intense exercise (IE) mice (TD+CUS+IE mice; n = 10). As our previous study [16] indicated that mice fed a normal diet with exposure to CUS showed depressive behavior, in this study, we omitted the experimental condition concerning the mice fed a normal diet with exposure to CUS. The C and TD mice were housed in standard 15481974 mouse cages with five mice per cage. The TD+CUS, TD+CUS+ME and TD+CUS+IE mice were housed in standard mouse cages divided into six cells to reduce their living space and to decrease their daily activity [17]. The TD, TD+CUS, TD+CUS+ME and TD+CUS+IE mice were fed a tryptophandeficient (TD) powered diet (Oriental Yeast Co., Ltd., Japan). The C mice were fed a TD powered diet supplemented with tryptophan at 214 mg per 100 g of powder diet, equal to the tryptophan content in a standard animal diet. All diets were mixed with hot water, kneaded, cut and dried to make hard pellets. Tap water was given to all mice. All mice were introduced to their respective diets for a week before the start of the CUS procedure. During the experimental period, all mice were allowed to eat and drink ad libitum. The weight of all mice was measured once a week throughout the experimental period.5. Behavioral Tests(1) Forced swimming test (FST). All mice were subjected to FST to evaluate depression-like behavior according to the method of Porsolt et al. [19]. For testing, cylinders (height, 2 cm; diameter, 15 cm) filled with water (25uC) were used to make the mice swim or float without touching their hindlimbs or tail on the bottom of the cylinder. Each mouse was individually placed in the cylinder and its movements were recorded for 6 min using a video camera. Immobility time, when the mouse performed the minimal movement required to stay afloat, was measured to evaluate depression-like behavior during the latter four minutes of the test. (2) Sucrose preference test (SFT). At the end of 28 days of CUS, all mice were subjected to SFT to evaluate depression-like behavior according to the method of Sakata et al. [20]. In brief, after animals had been habituated to two water bottles for 3 days in their home cages, a free choice between plain water and 12. Chronic Unpredictable StressThe timeline of the various experimental procedures is shown in Fig. 1. After 7 days of acclimation to cage and diet, all mice wereFigure 1. Experimental procedures. Habituation: habituation to cage, food and treadmill running; CUS: chronic unpredictable stress; PAT: passive avoidance test; ORT: object recognition test; SFT: sucrose preference test; FST: forced swimming test. doi:10.1371/journal.pone.0066996.gExercise Prevents Depression in TD MiceTable 1. Protocol of chronic unpredictable stress.Day time stress Immobilization; 3 h Cold isolation(uC); 3 hTimes 6Overnight stress Light on overnigh Web bedding overnigh Crowding overnight Food/water deprivation overnight Tilt of cage Stroboscope overnight TotalTimes 5 4 4 4 5 5Cage rotation (100 rpm); 3 h 5 Swim in water(18uC); 5 min 3 Rat odor; 3 hConfrontation with rat; 3 h 5 Totaldoi:10.1371/journal.pone.0066996.tsucrose solution was provided to each mouse. The positions of the bottles were counterb.