Sexually inexperienced, gonadally-intact male mice (10–19 weeks) were employed in this study. mGluR7 KO and wild-type littermates were from heterozygous mating couples, produced by back-crossing the mGluR7 KO line lacking the first coding exon and neighboring intron of the mGluR7 gene [23,24] onto the C57BL/6N background (RRID:MGI:5705075) for at least 11 generations. All animal protocols were compliant with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Animal Research, Shiga University of Medical Science. Animals were euthanized by cervical dislocation or CO2 inhalation following approved protocols.
All mice tested in behavioral studies were maintained on a 12/12 h light/dark cycle (light off at 8 PM). Food and water were provided ad libitum. Animals were identified by arbitrarily assigned numbers, such that the investigators performing the experiments were blinded to genotype. Additionally, the order of the experiments was changed each day.
Spinal transection (spinalization)
Spinalization was accomplished as in previous reports [25,26]. Under anesthesia with isoflurane, the spinal cord was completely transected intervertebrally using microscissors inserted between the 9th and 10th thoracic vertebrae. After filling the space of the removed spinal cord with Gelfoam (Pfizer, Washington, DC, USA), the incised site was sutured. As spinalization caused loss of control of urination, the Credé maneuver  was applied 3 times a day (early morning, early afternoon, and late afternoon) to assist voiding, i.e. gentle pressure was applied to the bulging bladder to cause urination. Bladders were emptied manually until a spontaneous return of micturition. Complete transection was confirmed by flaccid hindlimbs and post-mortem examination of the lesioned area stained with neutral red.
Penile responses during manual urination
During manually-assisted urination in the morning (when the bladder was full), penile responses (n = 9 for each genotype) were observed for 3 consecutive days. Before voiding, the bladder was palpated and urinary retention was estimated, and only those mice with full bladders were included. Counting started when urine began to flow and was observed for 90 sec. We defined penile responses according to 2 distinct categories as follows [28,29]:
1. flips: dorsiflexions of the penis due to a straightening of the penile body.
2. cups: intense erections with a flaring of the engorged glans penis.
The number of flips and cups was recorded. Additionally, the presence or absence of seminal emissions (materials) during manual urination was noted.
Penile reflex test
On day 1 (6 hrs after spinalization) and day 2, penile reflexes (n = 9 for each genotype) were assessed in a manner similar to a previous report . Briefly, the mice were placed on their backs, restrained by strips of adhesive tape across the midsection and at the base of the hind legs, and the glans penis was extracted as far as possible. Adhesive tape was placed at the base of the sheath to maintain exposure of the glans. Flips and cups were recorded. Testing ended 10 min after retraction of the penile sheath. Prior to thoracic transection, mice were restrained using a 50 ml conical tube and adhesive tape, and the glans penis was extracted. No flip or cup was observed in spinally-intact mGluR7 KO or wild-type mice.
All mice were tested on the 5th day post-transection according to a previous report . The effects of administration of 2.5 mg/kg, i.p. clonidine (Sigma, St-Louis, MO, USA) were examined. Three hrs prior to drug administration, the bladder was manually voided and the penis was examined immediately prior to testing to confirm the absence of seminal fluids or plugs. Black paper was placed in the cage to facilitate the observation of seminal emissions. The ejaculatory response was observed every 10 min for 30 min after clonidine administration by checking the coagulated seminal materials retrieved from the paper or from the shaft of the penis. Seminal emissions (materials) were collected, allowed to dry on a filter paper and weighed.
Wild-type and mGluR7 KO mice were anesthetized with medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol (5 mg/kg), and then perfused through the left ventricle with formaldehyde (4%; freshly depolymerized from paraformaldehyde) in 0.1 M sodium phosphate buffer (pH 7.4), after which the spinal cords were removed. These were then saturated with 30% sucrose/0.1 M sodium phosphate buffer at 4ºC, and cut into 35-µm coronal sections using a cryostat (CM3050 S; Leica Microsystems); these sections were treated as free-floating. Because we found that immunostaining with anti-mGluR7 antibody (07-239, Millipore; RRID:AB_310459) yielded a high background, we preabsorbed the antibody solution with excess mGluR7 KO brain sections to prevent non-specific immunoreactivities, as previously reported .
For triple-immunofluorescence histochemistry, some sections were incubated with a mixture of rabbit anti-mGluR7 antibody (1:6,000 final dilution in Can Get Signal immunostain Solution A), rat anti-5-HT antibody (clone YC5/45, MAB352, Sigma-Aldrich; 1:75; BRID:AB_11213564), and mouse anti-neuronal nitric oxide synthase (nNOS; A-11, sc-5302, Santa Cruz Biotechnology; 1:8,000 dilution; RRID:AB_626757) antibody. Sections were then treated with a mixture of Alexa Fluor 488-labelled goat anti-rabbit IgG (Life Technologies), Alexa Fluor 594-labelled goat anti-guinea pig IgG (Life Technologies), and Alexa Fluor 647-labelled goat anti-mouse IgG (Life Technologies) secondary antisera, as previously described . Some sections were incubated with a mixture of rabbit anti-mGluR7 antibody, guinea pig anti-galanin antibody (Peninsula Laboratories, Inc; 1:16,000 dilution; RRID:AB_518351), and mouse anti-nNOS antibody. The sections were then treated with a mixture of Alexa Fluor 488-labelled goat anti-rabbit IgG (Life Technologies), goat anti-guinea pig Alexa Fluor 594-labelled goat anti-guinea pig IgG (Life Technologies), and Alexa Fluor 647-labelled goat anti-mouse IgG (Life Technologies) secondary antibodies, and lastly, examined under a confocal laser scanning microscope (LSM780, Zeiss) using appropriate filters.
Immuno-histochemical analysis was performed as described previously . Briefly, the sections were incubated sequentially with (1) rabbit anti-mGluR7 antibody (1:12,000 final dilution in Can Get Signal immunostain Solution A), (2) 10 μg/ml goat biotinylated anti-rabbit IgG antibody (Vector Labs., Burlingame, CA), and (3) avidin-biotinylated peroxidase complex (ABC Standard, Vector Labs., Burlingame, CA; 1:100 dilution); all incubation media were prepared using 25 mM phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (PBS-Tx). Bound peroxidase was visualized by incubation with 0.02% diaminobenzidine tetrahydrochloride (DAB), and 0.003% H2O2 in 50 mM Tris-HCl (pH 7.6).
Behavioral data were analyzed by two-way analysis of variance (ANOVA) for repeated measurements, or unpaired t test for independent samples. Some data (in which variances were not homogeneous between genotype groups) were analyzed by nonparametric tests (Mann-Whitney U test).