Drug Treatment

Male C57BL/6J mice were singly housed from the age of 8 weeks in the institutional standard condition (14:10 light/dark cycle; lights on at 6:00 A.M. through 8:00 P.M.) at 23 ± 1°C with food and water available ad libitum. Following 1 week of acclimation, fluoxetine hydrochloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was orally applied at a dose of 14 or 22 mg/kg/day. In order to minimize stress associated with drug administration, fluoxetine was dissolved in the drinking water. The fluoxetine solutions were prepared everyday, and concentrations of fluoxetine were determined for individual mice based on the water consumption during preceding 24 h and the body weight measured every other day [9]. Saccharin (0.2%) was included in the fluoxetine solution to keep water intake comparable to the baseline [9]. Control mice were given water with or without saccharin, and all data were pooled. After 4 weeks of fluoxetine treatments, behavioral tests were started, and fluoxetine treatments were continued during the tests. The 5-HT 4 receptor heterozygous mutant mice (strain name: B6.129P2-Htr4 < tm1Dgen >/J) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). These mice had been backcrossed to the C57BL/6J background for 10 generations. Male wild-type (5-HT 4 +/+) and homozygous mutant (5-HT 4 -/-) littermates from heterozygous mating were treated with fluoxetine and used for experiments. There was no significant difference in the actual doses of fluoxetine administered to mice between the genotypes. All procedures were approved by the institutional Animal Care and Use Committee.

Home cage activity monitoring

Mice were singly housed in the cage (15 × 25 × 30 cm) equipped with an infrared video camera at the top, and locomotor (horizontal) activity in the cage was continuously monitored. Outputs from the cameras were fed into a personal computer. Images were captured at a rate of one frame per second, and the distance traveled per minute was analyzed online using software based on the public domain ImageJ (ImageJ HC8; O'Hara and Co., Ltd., Tokyo, Japan). Since activity of mice during the light period could be affected by external stimuli such as noise made by workers in the animal facility, the activity during the dark period (nocturnal activity) was evaluated in quantitative analyses.

Behavioral experiments

Mice were transferred to a behavioral testing room and allowed to acclimatize to the environment of the room for at least 1 h 30 min before starting behavioral tests. All tests were performed between 13:30 P.M. and 18:00 P.M. The tests were sequentially performed on different days in the order of the open-field test, light-dark transition test, elevated plus-maze test, forced swim test and tail suspension test. Only one test was conducted for each mouse in a day. Room temperature was kept at 24 ± 0.5 °C. To minimize olfactory cues from the previous trial, each apparatus was wiped and cleaned with a hypochlorous acid solution (~15 ppm, pH 5 - 6.5) before each test except the forced swim test and the tail suspension test.

The open-field test was carried out to examine locomotor activity and anxiety-related behavior in a novel environment. The open-field apparatus composed of opaque white walls and a floor (50 × 50 × 50 cm) was illuminated at an intensity of 40 lux. Each mouse was placed in the center of the open-field arena, and then locomotor (horizontal) activity was monitored for 20 min via a CCD camera positioned above the apparatus. The ambulatory distance and relative time spent in the central zone were measured. To calculate relative time spent in the center, the floor of the apparatus was divided into 25 squares and time spent in the central nine squares was measured. Fluoxetine did not significantly affect vertical activity assessed by measuring the number of infrared photobeam interruption. All records were stored on a PC and analyzed using software based on the public domain ImageJ (ImageJ OF; O'Hara and Co., Ltd., Tokyo, Japan).

The light/dark transition test was carried out to assess anxiety-related behavior. The apparatus for this test was composed of a box (21 × 42 × 25 cm) divided into two compartments of equal size. One compartment was brightly illuminated at an intensity of 600 lux and a CCD camera was equipped on the ceiling, whereas the other was not illuminated (8 lux) and an infrared camera was equipped on the ceiling. The partition dividing the compartments had an opening (5 × 3 cm) with a sliding door through which mice could move from one compartment to the other. Single mice were put into the dark compartment and the sliding door was automatically opened after 5 s. The behavior of mice was monitored for 10 min. The total number of transitions between chambers, time spent in each side, the first latency to enter the light side and the distance traveled in each side were recorded automatically and analyzed using software based on the public domain ImageJ (ImageJ LD2, O'Hara & Co., Ltd., Tokyo, Japan). When mice did not enter the light compartment, the latency was considered 600 s.

The elevated plus-maze test was carried out to examine anxiety-related behavior. The apparatus consisted of a central platform (5 × 5 cm), two opposed open arms (25 × 5 cm) and two opposed closed arms of the same size, but with 15-cm-high opaque walls. The edges of the open arms were raised 0.25 cm to avoid falls of mice. The floor of each arm was made of white plastic. The apparatus was elevated to a height of 50 cm above the floor and illuminated at an intensity of 40 lux. At the beginning of each test, one of the open arms was blocked by a transparent obstacle and each mouse was placed on the central platform, facing the blocked open arm. After the mouse entered either of the closed arms, the block was removed and the test was started. Activity of mice was monitored for 10 min via a CCD camera positioned above the apparatus. The time spent in each arm, the number of entries into each arm and the ambulatory distance were recorded and analyzed using software based on the public domain ImageJ (ImageJ EP, O'Hara & Co., Ltd., Tokyo, Japan).

The forced swim test was carried out to assess depression-related behavior. The apparatus consisted of a transparent plastic cylinder (22 cm in height and 12 cm in diameter) placed in a box (30.5 × 40 × 40 cm). The cylinder was filled with water (25°C) up to a height of 10 cm and was illuminated from the bottom of the box. Each mouse was placed into the cylinder and activity was monitored for 15 min via a CCD camera mounted on the top of the box. Immobile time except for first 2 min was measured. The cylinder was refilled with clean water after each test. All records were stored on a PC and analyzed using software based on the public domain ImageJ (ImageJ PS4, O'Hara & Co., Ltd., Tokyo, Japan).

The tail suspension test was carried out to examine depression-related behavior. The tip (1 cm) of mouse tail was securely fastened with adhesive tape to a metallic plate. The plate was hung from the ceiling of a test box (30.5 × 40 × 40 cm). Immobile time was measured for 6 min with a CCD camera mounted on the side of the box. All records were stored on a PC and analyzed using software based on the public domain ImageJ (ImageJ PS4, O'Hara & Co., Ltd., Tokyo, Japan).

Electrophysiology

Mice were decapitated under deep halothane anesthesia and both hippocampi were isolated. Transverse hippocampal slices (380 μm) were cut using a tissue slicer in ice-cold sucrose-containing saline composed of (in mM): sucrose 72, NaCl 80, KCl 2.5, NaH 2 PO 4 1.0, NaHCO 3 26.2, glucose 20, CaCl 2 0.5, MgCl 2 7 (equilibrated with 95% O 2 /5% CO 2 ). Slices were then incubated for 30 min at 30°C and maintained in a humidified interface holding chamber at room temperature (24 - 27°C) before recordings. Electrophysiological recordings were made in a submersion-type chamber maintained at 27.0 - 27.5°C and superfused at 2 ml/min with saline composed of (in mM): NaCl 125, KCl 2.5, NaH 2 PO 4 1.0, NaHCO 3 26.2, glucose 11, CaCl 2 2.5, MgCl 2 1.3 (equilibrated with 95% O 2 /5% CO 2 ). Field excitatory postsynaptic potentials (fEPSPs) arising from the mossy fiber synapses were evoked by electrical stimulation delivered to the dentate granule cell layer and recorded from the stratum lucidum of CA3 using a glass pipette filled with 2 M NaCl. The amplitude of fEPSPs was measured on analysis as described [28]. A criterion used to identify the mossy fiber input was more than 85% block of fEPSP amplitude by an agonist of group II metabotropic glutamate receptors, (2 S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (1 μM) (Tocris Bioscience, Bristol, UK). Single electrical stimulation was delivered at a frequency of 0.05 Hz for baseline recordings and frequency facilitation was examined at 1 Hz. All recordings were made using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), filtered at 2 kHz and stored in a personal computer via an interface (digitized at 5 - 10 kHz).

Statistics

The number of data (n) represents the number of mice. Since some data from drug-treated mice did not distribute normally, nonparametric tests were used to evaluate statistical significance. The two-tailed Mann-Whitney test and Dunn's multiple comparison test were used to compare two groups and three groups or more, respectively, with the significance level P < 0.05. All statistical tests were performed using GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA, USA).