All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Sugitani Campus of the University of Toyama. All protocols were approved by the Committee for Animal Care and Use of the Sugitani Campus of the University of Toyama. The approval number for the animal experiments is A2011-INM1. All efforts were made to minimize the number of animals used.

Materials

Diosgenin (Wako, Osaka, Japan) was dissolved in ethanol at 10-fold the final concentration and the stock solution was diluted in a 5% glucose aqueous solution. The vehicle solution was 10% ethanol in 5% glucose. A specific neutralizing antibody for 1,25D 3 -MARRS (clone Ab099) was provided as a gift by Dr. Nemere.

Animals

Male ddY mice (6 weeks old, Japan SLC, Shizuoka, Japan) were housed with free access to food and water and kept in a controlled environment (22 ± 2°C, 50 ± 5% humidity, 12-h light cycle starting at 7:00 am). The drug or vehicle solution was intraperitoneally administered once a day for 5 days (Figure 1B), 6 days (Figure 3) or 7 days (Figure 1A).

Open field test

On the day before a training session, mice were individually habituated to an open-field box (30 cm × 40 cm; height, 36.5 cm) for 10 min and their paths were tracked by a digital camera. The distance moved for 10 min was analyzed using the locomotion activity function of EthoVision 3.0 (Noldus, Wageningen, Netherlands). Testing was carried out in a dimly illuminated room (90 lux).

Object recognition test

Two identical objects (colored ceramic ornaments) were placed at a fixed distance within a square box (30 cm × 40 cm; height, 36.5 cm, 90 lux). A mouse was then placed at the center of the box and the number of times it made contact with the two objects was recorded during a 10-min period (training session). Mice were then placed back into the same box 48 h after the training session and one of the objects used during the training session was replaced with a novel object (another ceramic ornament with a different shape and color). The mice were then allowed to explore freely for 10 min; the number of times they made contact with each object was recorded (test session). A preference index, defined as the ratio of the number of times a mouse made contact with any of the objects (training session) or the novel object (test session) over the total number of times the mouse made contact with both objects, was used to measure cognitive function for objects.

Immunohistochemistry

Precisely 60 min after the novel object recognition test session, mice were anesthetized and transcardially perfused with cold physiological saline. The brains were carefully removed from the skull, immediately immersed in 30% sucrose-PBS and stored at −30°C. The brains were cut into 20 μm coronal slices every 100 μm in the medial prefrontal cortex area (bregma + 1.70 to + 2.46 mm) and the perirhinal cortex area (bregma −1.34 to −2.06 mm) using a cryostat (CM3050S, Leica, Heidelberg, Germany). The slices were fixed with 4% paraformaldehyde and stained with a monoclonal antibody against pNF-H (1:500) (Covance, Emeryville, CA, USA) and polyclonal antibody against c-Fos (1:500) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C for 20 h. An Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300) and Alexa Fluor 568-conjugated goat anti-rabbit antibody (1:300) were used as secondary antibodies (Molecular Probes, Eugene, OR, USA). For staining c-Fos, NeuroTrace Fluorescent Nissl Stains (Molecular Probes) was used as counter staining to confirm its neuron-specific expression. The fluorescent images for axons and c-Fos were captured using a fluorescent microscope (BX-61, Olympus, Tokyo, Japan) at 324 μm × 430 μm. Six successive brain slices from the medial prefrontal cortex and six successive slices from the temporal cortex containing the perirhinal cortex were captured for quantification. The lengths of pNF-H-positive axons were measured using an image analyzer Neurocyte (Kurabo, Osaka, Japan), which automatically traces and measures neurite length without measuring cell bodies. The area of c-Fos-positive staining was measured using the Image J (http://rsbweb.nih.gov/ij/).

In vivo single unit recordings in the mPFC and hippocampal CA1

C57BL/6 mice (8 weeks old, male) were i.p. administered diosgenin for 21 days and were anesthetized with i.p. injection of urethane (1.5 g/kg; Sigma-Aldrich, Oakville, ON, Canada) and mounted on the stereotaxic apparatus. Body temperature of the animals was maintained with the heat pad during recordings. After incision was given to the skull skin, burr holes were made onto the skull by the dental drill for placements of recording electrodes. Extracellular electrodes were made of 1 mm O.D. Omegadot borosilicate glass tubes (WPI, Saratota, FL, USA) pulled with the puller. Typical tip resistance of electrodes filled with 2 M NaCl solution was approximately 3 ~ 5 MΩ. One recording electrode was lowered into the right hemisphere of the layer V–VI of the mPFC (+1.5 mm A/P, +0.4 mm M/L and −2.8 mm D/V). The other recording electrode was aimed at CA1 (−2.3 mm A/P, +1.7 mm M/L and −1.5 mm D/V). Five minutes of single unit recordings were obtained from each animal. Extracellular single unit signals were 10,000 times amplified and band-pass filtered at 0.1–10 kHz with the amplifiers. Analog signals were then digitized with the digitizer and stored in the computer for off-line analysis. Correlation of single unit discharges between the mPFC and CA1 were assessed by coherence analyses, using the functions implemented in the statistical analysis software STATISTICA (StatSoft, Tulsa, OK, USA). All animals were killed rapidly on completion of electrophysiological recordings with an overdose of pentobarbital (100 mg/kg, i.p.) and after cryoprotection of brains in 30% sucrose solution, brains were sectioned at 30 μm thickness with the sliding microtome. Recording and stimulation sites were confirmed with Nissl staining using the light microscope.

Primary culture

Embryos were removed from pregnant ddY mice (Japan SLC) at 14 days of gestation. The cortices were dissected and the dura mater was removed. The tissues were minced, dissociated and cultured with neurobasal medium (Invitrogen, Grand Island, NY, USA) that included B-27 supplement (Invitrogen), 0.6% D-glucose and 2 mM L-glutamine at 37°C in a humidified incubator with 10% CO2 on 8-well chamber slides (Falcon, Franklin Lakes, NJ, USA) coated with 5 μg/ml poly-D-lysine. The seeding cell density was 2.9 × 104 cells/cm2.

Measurement of axonal density

Three days after cell seeding, the control antibody (normal rabbit IgG, 1:500) or polyclonal anti-rabbit 1,25D 3 -MARRS (Ab099 clone, 1:500) was applied to the cells. After a 15-min incubation with the antibody, the cells were treated with vehicle solution or diosgenin (1 μM). The cells were fixed with 4% paraformaldehyde and immunostained at 4°C for 20 h with a monoclonal antibody against pNF-H (1:500) as an axonal marker and a polyclonal antibody against MAP2 (1:500) as a neuronal marker. Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300) and Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:300) were used as secondary antibodies. The fluorescent images were captured using a fluorescent microscope system (BX61/DP70, Olympus) at 324 μm × 430 μm. Twenty-one images were captured per treatment. The lengths of the pNF-H-positive axons were measured using the Neurocyte image analyzer (Kurabo). The sum of the axon lengths was divided by the number of MAP2-positive neurons in an image. The resulting axon density was averaged over all of the images.

Surgical procedure

Mice were anesthetized with chloral hydrate (500 mg/kg) and positioned in a stereotaxic apparatus. The scalp was shaved and cut and the skull was exposed. A cannula (Brain Infusion Kit 3, Alzet, Cupertino, CA, USA) was positioned into a lateral ventricle at the following coordinates: −0.22 mm A/P, +1.0 mm M/L and −2.5 mm D/V. The free end of the cannula was connected to a mini-osmotic pump (Alzet, Model 1007D) via a 3.5-cm piece of polyvinylchloride (PVC) tubing (Alzet). The mini-osmotic pump and connecting PVC tubing were filled with artificial cerebrospinal fluid (aCSF) or anti-1,25D 3 -MARRS dissolved in aCSF. The CSF production rate in a mouse is 18 μl/h. The infusion rate of the mini-osmotic pump was 0.5 μl/h. Therefore, the anti-1,25D 3 -MARRS antibody and aCSF were mixed at 1:14 ratio to reach a 1:500 dilution, which is the effective dose of the antibody for neutralization. The filled pumps were incubated in sterile saline at 37°C for at least 16 h before being implanted under the dorsal skin of the mouse's back. The cannula base and attached piece of PVC tubing were fixed to the skull with Loctite cyanoacrylic 454. During and after surgery, mice were placed on a heating pad to maintain body temperature.

Statistical analysis

Statistical comparisons were performed using one-way analysis of variance (ANOVA) with post hoc Dunnett's tests, unpaired t-tests and paired t-tests in GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered significant. The mean values of the data are presented together with the SE.