TMP, reserpine, and clomipramine hydrochloride were

purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and fluoxetine (FLU) hydrochloride was purchased from Eli Lilly and Company Limited (USA). 5-Hydroxytryptophan (5-HTP) was purchased from Jinjinyao Amino Acid Co. Ltd (Tianjin, China), and NE bitartrate injection purchased from Grass Plant Technology Co. Ltd. (Xian, China).

Special pathogen free (SPF) adult male Sprague-Dawley (SD) strain rats weighing 180 - 220 g and Kunming (KM) mice weighing 18 - 22 g purchased from the Experimental Animal Center of Guangdong Medical College (experimental animal license SCXKyue 2007-2008A034, No.0001909; Zhanjiang, China), were used across all the experiments. They had free access to tap water and standard laboratory food unless otherwise stated. Housing conditions were controlled, temperature was maintained at 22˚C ± 1˚C with approximately 60% relative humidity. They were kept on a reversed 12/12 h light/dark cycle (light 07:00 - 19:00 h). Animals were acclimated to the animal quarters for 1 week (for rats) or 3 days (for mice) before any experimental procedure. All the animals were treated in compliance with “Guidance Suggestion for the Care and Use of Laboratory Animals” issued by The Ministry of Science and Technology of People’s Republic of China.

Measurement of immobility time was carried out by observing the motoric activity of the mice or rats, which were placed in a pool of water. A glass cylinder, 15 cm (for mice) or 30 cm (for rats) in diameter, height 20 cm (for mice) or 40 cm (for rats), was filled with water to a height of 12 cm (for mice) or 24 cm (for rats). The temperature of water was 25˚C ± 1˚C. Each mouse or rat received twice a respective dose of FLU (20 mg/kg, ig) or TMP (20, 40, 80 mg/kg, ig) 17 h and 7 h (for FLU) or 30 (for rats) or 10 (for mice) min (for TMP) before the forced-swimming test. This dose of FLU was chosen because it had been shown to be differentially effective in HR vs LR animals [9]. The mice or rats in controls received ip injection of physiological saline instead of TMP. And then the animals were subjected to the tests. Measurement was carried out for six (for mice) or five min (for rats). For mice, the first two min the animal was allowed to adjust to the new conditions; after these two min, the immobility time that alternated with conditions of enhanced motor activity was measured. Immobility time was measured with a stopwatch for the next four min. For rats, no time the animal was allowed to adjust to the new conditions. Immobility time is the time during which the animal floated on the surface with front paws together and made only those movements which were necessary to keep afloat. Shorter immobility time is an indicator of the stronger antidepressant effect of the tested substance [10-12].

The tail suspension test was based on the method of Steru et al. [13]. Each mouse received twice a respective dose of FLU (20 mg/kg, ig) or TMP (20, 40, 80 mg/kg, ip) 17 h and 7 h (for FLU, ig) or 10 min (for TMP, ig) before the tail-suspension test, the mice in control received ig physiological saline instead of TMP, and the animals were subjected to the test 10 min later. A cord of about 50 cm in length was stretched between two metal tripods at a height of ca 70 cm, to which the mice were attached by the tail with sticky tape. After the initial period (the first two min) of vigorous motor activity, the mice became still and the immobility time was measured with a stopwatch for a total duration of 4 min [13,14]. Mice were considered immobile when they hung passively and completely motionless.

Mice were assigned randomly into six matched groups (each consisting of 12 animals) based on rectal temperature (for hypothermia test) or body weight (for akinesia and ptosis tests) before onset of the test. Group1 (control) was treated with physiological saline, and maintained under identical conditions to other groups for use as control. Group 2 (reserpine) was given reserpine alone (2 mg/kg, ip). Group 3 (clomipramine) was given both clomipramine hydrochloride (40 mg/kg × 2) and reserpine (2 mg/kg, ip). Group 4, 5, 6 (TMP) were given both TMP (20, 40, 80 mg/kg × 2) and reserpine (2 mg/kg, ip), respectively. Clomipramine hydrochloride or TMP were administered ig twice 24 h and 30 min prior to reserpine, and then the mice were treated with reserpine.

At 60 min after the injection of reserpine the mice were placed in a circle 7.5 cm in diameter to observe for 30 s, the number of mice out of circle in each group was calculated [15]. The degree of ptosis of each animal was recorded at 90 min after the injection of reserpine. For the evaluation of ptosis, mice were placed on a shelf (20 cm above the bench top). The degree of ptosis was rated according to the following rating scale: 0, eyes open; 1, one-quarter closed; 2, half closed; 3, three-quarters closed; and 4, completely closed [2,16]. The rectal temperature of each animal was measured with an electrical thermometer at 2, 4, 6 h after the injection of reserpine [4,17].

Mice were assigned randomly into six matched groups (each consisting of 12 animals) based on body weight before onset of the test. Group 1 (control) was treated with physiological saline, and maintained under identical conditions to other groups for use as control. Group 2 (NE) was given NE bitartrate alone (4.0 mg/kg, ip). Group 3 (clomipramine) was given both clomipramine (40 mg/kg × 2) and NE bitartrate (4.0 mg/kg, ip). Group 4, 5, 6 (TMP) were given both TMP (20, 40, 80 mg/kg × 2) and NE bitartrate (4.0 mg/kg, ip), respectively. Clomipramine or TMP were administered ig twice 24 h and 30 min prior to NE bitartrate, and then the mice were treated with NE bitartrate. Lethality (number of mice which died/total number of mice) was recorded and calculated 48 h after the injection of NE bitartrate.

Results were expressed as the mean ± standard deviation (SD) unless otherwise stated. The data were analyzed by one-way ANOVA, followed by Dunnett’s t-test. Comparisons of lethality and akinesia among groups were made according to χ²-test. Signicant difference was determined when P < 0.05.

Figures 2 and 3 show the effects of TMP and FLU on the duration of immobility in the forced swim tests. When compared with the control group, TMP at the doses of 20, 40, and 80 mg/kg reduced the duration of immobility by 11.5%, 15.5% and 24.0% (P < 0.05)(for rats), and 21.5%, 29.7%, and 39.9%(P < 0.05) (for mice), respectively. Similar effects were observed for treating the rats and mice with classical antidepressant FLU at the dose of 20 mg/kg which served as a positive control of the experiment. The reduction in the duration of immobility given with FLU 20 mg/kg) was 34.1% (for rats) and 37.0% (for mice), respectively. TMP at the dose of 80 mg/kg was as effective as the positive control (FLU, 20 mg/kg) in these tests.

Figure 4 shows the effects of TMP and FLU on the duration of immobility in the tail suspension test. TMP at the doses of 40 and 80 mg/kg significantly reduced the dura-