Animals

We used Glra4 mutant mice, harbor an 11-bp deletion in exon 4, prepared as described previously [14]. To avoid potential off-target effects, the mutant mice were backcrossed with C57BL/6J mice for at least 4 generations. Because Glra4 is located on the X chromosome [19] and male and female mice have different doses of the gene, same-sex mice were used for all experiments. Hemizygous mutant males (Glra4−/Y) and their WT (Glra4+/Y) littermates were used for the behavioral analyses, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting experiments. The mice were maintained under a 12:12-h light:dark cycle at 22 ± 2 °C and relative humidity of 40% to 60% and with ad libitum access to chow (CE2, CLEA Japan, Inc.; CRF-1, Oriental Yeast Co., LTD., Tokyo, Japan) and water.

RNA isolation and cDNA synthesis

RNA isolation and subsequent RT-qPCR reactions were performed according to the MIQE guidelines [20]. Mice were decapitated, and brains were collected intact or dissected into different regions according to experiment purpose and rapidly frozen in liquid nitrogen. Total RNA was extracted from mouse brain tissues using TRIzol (Invitrogen; Carlsbad, CA, USA) following the manufacturer’s protocol. The RNA quality and quantity were assessed on a BioSpec-nano spectrophotometer (Shimadzu, Kyoto, Japan) and the A260/A280 ratio was confirmed to be 1.9–2.2. All extracted RNA samples were treated enzymatically by DNAse I (Turbo DNAse; Ambion Inc., Austin, TX, USA) and 1 µg RNA was used to synthesize complementary DNA (cDNA) using the PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa Bio Inc.) according to the manufacturer's protocols. The cDNA template was diluted 1/20 for further use in RT-qPCR.

RT-qPCR

Primers were designed using the PrimerQuest® Tool and Primer3 software. The primer pairs used in this study are shown in Additional file 1: Table S1. The optimum annealing temperature for all primers was tested using gradient PCR. Primer specificity was confirmed by the appearance of a single band on the gel and a single peak on the melting curve of the qPCR reaction. The efficiency of each set of primers was assessed by RT-qPCR on serial dilutions of cDNA from brain tissues and was confirmed to be in the range of 90% to 110%. PCR was performed in a 20 µl volume containing 2 µl template, 1 µl of 10 µM of each primer, 0.2 µl Phusion DNA Polymerase, 4 µl of 5 × Phusion HF Buffer (New England Biolabs, Ipswich, MA, USA), and 2 µl of 10 mM dNTPs with the following thermal cycler conditions: 98 °C for 30 s, 30 cycles of 98 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s, and then 72 °C for 5 min for the final extension. Each biological sample had 3 or 4 technical replicates and the mean of the triplicates or quadruplicates were taken to be the Ct representing the biological sample. The Ct values of the housekeeping genes were used to normalize target genes and the fold change (log2) for each sample was calculated relative to the reference sample using the 2−ΔΔCt method.

Western blotting

Brain samples were collected, frozen immediately in liquid nitrogen, and stored at − 80 °C until use. The samples were homogenized in ice-cold RIPA lysis buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 0.1% Triton X100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl-sulfate) supplemented with 1 protease inhibitor tablet (cOmplete™, Mini Protease Inhibitor Cocktail; Roche, Basel, Switzerland) using a sonicator. The lysates were centrifuged at 14,000 rpm for 20 min at 4 °C and the supernatants were stored at − 30 °C until use or used immediately for protein quantification with a protein assay bicinchoninate kit (Nacalai Tesque, Inc., Kyoto, Japan). Equal amounts of lysates in each sample were mixed with 2 × Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for protein denaturation at 95 °C for 5 min. To detect proteolipid protein 1 (PLP1), the denaturation step was skipped because PLP1 aggregates in boiling lysates [21]. The lysates were then subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to transblot turbo polyvinylidene difluoride transfer packs using the Trans-Blot Turbo System (Bio-Rad Laboratories, Inc.). The membranes were then blocked in Tris-buffered saline with 0.1% Tween (TBST) containing 5% (w/v) non-fat milk for 1 h at room temperature to block nonspecific protein binding sites. The membranes were then incubated overnight with primary antibodies diluted in blocking buffer; rabbit polyclonal anti GlyR α4 (Cat. No. orb157164, 1:3000 dilution; Biorbyt, Cambridge, UK), rabbit mAb anti-PLP1 (Cat. No. ab254363, 1:2000 dilution, Abcam, Cambridge, UK), and rabbit mAb anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cat. No. 2118S, 1:5000 dilution; Cell Signaling Technology, Danvers, MA, USA). These antibodies had high specificity for the target proteins in mice. After washing 3 times with TBST for 5 min/wash, the membranes were incubated for 1 h at room temperature with anti-rabbit IgG, horseradish peroxidase-linked antibody diluted in blocking buffer (cat. no. 7074, 1:3000 dilution; Cell Signaling Technology). Membranes were again washed 3 times in TBST for 5 min/wash. To confirm the absence of short fragments of GlyR α4 in Glra4 mutant mice, the membrane was first incubated with anti-GlyR α4 antibody and then stripped with stripping buffer and re-probed with anti-GAPDH. Proteins bands were detected using Clarity Western ECL substrate (Bio-Rad Laboratories, Inc.), and then the blots signals were captured using a Luminescent Image Analyzer LAS-3000 (FUJIFILM, Tokyo, Japan).

Experimental design of behavioral analysis

Glra4−/Y mice (n = 18) and control (Glra4+/Y) mice (n = 14) were subjected to a comprehensive behavioral test battery [22]. The behavioral tests were conducted in the following order (see Table 1): general health and neurologic screening, light/dark transition, open field, elevated plus-maze, hot plate, social interaction test in a novel environment, rotarod, three-chamber social approach, startle response/prepulse inhibition, Porsolt forced swim, T-maze, Barnes circular maze, tail suspension, contextual and cued fear conditioning tests, and social interaction test in the home cage. Super hypochlorous water and 70% ethanol were used to clean each apparatus between animals to prevent bias due to olfactory cues. The interval between tests was at least 1 day. All behavioral testing was performed between 9:00 a.m. and 6:00 p.m. Raw data from the behavioral tests and information about each mouse are accessible on the public database “Mouse Phenotype Database” (http://www.mouse-phenotype.org/).

Table 1 Schedule and summary of the comprehensive behavioral analysis of Glra4 mutant miceGeneral health and neurologic screening

In the general health and neurologic screens, body weight, body temperature, and muscle strength were measured, and righting, whisker twitch, ear twitch, and key jangling reflexes were evaluated. In addition, the presence of whiskers or bald hair patches was recorded. Neuromuscular strength was assessed using the wire hang and grip strength tests. In the wire hang test, each mouse was placed on a wire mesh, and the latency to fall after being inverted was recorded with a 1-min cut-off time. Forelimb grip strength was assessed using a grip strength meter (O’Hara & Co., Tokyo, Japan). Mice were lifted and held by the tail so they would grasp a wire grid using their forepaws. The mice were then gently pulled backward by the tail until they released the grid. The peak force applied by the forelimbs of each mouse was recorded in Newtons (N). For each mouse, the test was repeated 3 times and the largest value was used for statistical analysis.

Light/dark transition test

The light/dark transition test was performed as previously described [23]. The apparatus consisted of a cage (21 × 41.5 × 25 cm) divided into 2 sections of equal size by a partition with a door (O’Hara & Co.). One chamber was dark (< 5 lx), while the other was brightly illuminated (~ 390 lx). Mice were placed into the dark chamber; the door was opened after 3 s and the mice were allowed to move freely between the chambers for 10 min. The following parameters were recorded automatically using ImageLD software (see section, “Image analysis”): total number of transitions between chambers, time spent in each chamber (s), latency to first enter the light chamber (s), and distance traveled in each chamber (cm).

Open field test

The apparatus comprised a square cage (42 × 42 × 31 cm; Accuscan Instruments, Columbus, OH, USA) illuminated at 100 lx. Each mouse was placed in the corner of the open field apparatus and allowed to move freely while being recorded for 120 min. The following parameters were measured: total distance traveled (cm), vertical activity (rearing measured by counting the number of photobeam interruptions), time spent in the center area (s) (20 × 20 cm), and beam-break counts for stereotyped behaviors.

Elevated plus-maze test

The elevated plus-maze test was performed as previously described [24]. The apparatus consisted of 2 arms (25 × 5 cm) with 3 mm-high ledges along the sides and distal end (open arms) and 2 enclosed arms (25 × 5 cm) with 15-cm high transparent walls along the sides and distal end (closed arms) (O'Hara & Co.). Arms of the same type were arranged opposite each other, and the arms and the central square (5 × 5 cm) were made of white plastic plates. The maze was elevated 50 cm above the floor. Each mouse was placed in the central square of the maze, facing a closed arm, and was recorded for 10 min. The illumination level at the center of the maze was 100 lx. The following parameters were calculated automatically using ImageEP software (see section, “Image analysis”): percentage of entries into the open arms, time spent in the open arms (s), total number of entries, and total distance traveled (cm).

Rotarod test

The rotarod test was performed on rotating drums (3 cm diameter) using an accelerating rotarod (UGO Basile Accelerating Rotarod, Varese, Italy). The speed of the rotarod accelerated from 4 to 40 rpm over a 5-min period. The time each animal was able to maintain its balance on the rod was measured (s).

Startle response/prepulse inhibition test

The acoustic startle response/prepulse inhibition test was measured using a startle reflex measurement system (O’Hara & Co.) as previously described [25]. Each mouse was gently placed in a Plexiglas cylinder where it was left undisturbed for 10 min as a test session. White noise (40 ms) was used as the startle stimulus for all trial types and the startle response was recorded for 400 ms (measuring the response every 1 ms) starting with the onset of the startle stimulus. The background noise level in each chamber was 70 dB. The peak startle amplitude recorded during the 200-ms sampling window was used as the dependent variable. The intensity of the startle stimulus was 110 or 120 dB. The prepulse sound was presented 100 ms before the startle stimulus, and the intensity was either 74 or 78 dB. Four combinations of prepulse and startle stimuli were used (74, 110; 78,110; 74,120; and 78, 120 dB). Six trial types were used in each session (i.e., 2 types for startle stimulus-only trials, and 4 types for prepulse inhibition trials). Six sessions of the 6 trial types were presented in a pseudorandom order such that each trial type was presented once within a block. The average inter-trial interval was 15 s (range 10–20 s).

Tail suspension test

The tail suspension test was performed in white plastic chambers (33 × 56 × 45 cm) (O’Hara & Co.) where each mouse is suspended by its tail with adhesive tape 30 cm above the floor. The behavior of the mouse was recorded with a video camera for 10 min. The immobility time was measured using ImageTS software (see section, “Image analysis”). Immobility was defined as time spent not moving that lasted more than 2 s.

Porsolt forced swim test

The Porsolt forced swim test was performed in plastic cylinders (height 22 cm, inner diameter 11 cm) (O’Hara & Co.) were filled with super hypochlorous water (approximately 23 °C) to a height of 7.5 cm. The mice were placed in the cylinder and allowed to swim for 10 min; the immobility time and distance traveled were recorded using ImageTS software (see section, “Image analysis”). Immobility was defined as time spent not moving that lasted more than 2 s.

Social interaction test in a novel environment

The social interaction test in a novel environment was performed as previously described [25]. Two mice of the same genotype that had never before been exposed to each other were placed in a box (40 × 40 × 30 cm) and allowed to move freely for 10 min. Several parameters, such as number of contacts, number of active contacts, contact duration (s), total distance traveled (cm), and mean duration/contact, were analyzed using ImageSI software (see section, “Image analysis”). An active contact was defined as when the 2 mice traveled together for at least 10 min.

Three-chambered social approach test

The sociability and social novelty preference tests were performed as previously described [26, 27]. The apparatus consisted of a rectangular, 3-chamber lidded box with a video camera (O’Hara & Co.). The 3 chambers (each 20 × 40 × 46.5 cm) were arranged side-by-side, each pair separated by a dividing wall with a small square opening (5 × 3 cm) to allow the mice to freely navigate among the chambers. In this test, unfamiliar mice that had no earlier contact with the test mice were enclosed in round cages (height 11 cm, bottom diameter 9 cm, vertical bars 0.5 cm apart) that allowed nose contact but prevented aggressive behavior. An unfamiliar C57BL/6 J male mouse (stranger 1) in a round cage was placed in 1 of the side chambers, an empty round cage was placed in the same relative location in the other side chamber, and the test mouse was placed in the middle chamber and allowed to explore the entire social test box for 10 min. The time the test mouse spent in each chamber was measured to quantify sociability for the first stranger compared with the empty cage. For the next session, another unfamiliar mouse (stranger 2) was placed in the chamber that was empty during the first 10-min session. The test mouse was then placed in the middle chamber and allowed to explore for 10 min and to choose between the first, already-investigated unfamiliar mouse (stranger 1) and the novel unfamiliar mouse (stranger 2). The time spent in each chamber during the second 10-min session was measured to quantify social novelty preferences. Data acquisition and analysis were performed automatically using ImageCSI software (see section, “Image analysis”).

Home cage social interaction test

Home cage social interaction monitoring was performed as previously described [26]. The system comprised a home cage (29 × 19 × 13 cm) and a filtered cage top containing an infrared video camera. Two mice of the same genotype, which had been housed separately, were placed together in the home cage. Their social interaction and locomotor activity were monitored for 1 week. Social interaction was measured by counting the number of particles detected in each frame: 1 particle indicates that the 2 mice are close to each other whereas 2 particles indicate that the 2 mice are apart from each other. The analysis was performed automatically using ImageHC software (see “Image analysis”).

Barnes maze test

The Barnes circular maze test was conducted on a white circular surface, 1.0 m in diameter, with 12 holes equally spaced around the perimeter (O’Hara & Co.). A black Plexiglas escape box (17 × 13 × 7 cm) containing paper cage bedding was located under 1 of the holes. For each mouse, the location of the target was consistent but randomized across mice. The maze was rotated after each trial, and to prevent bias based on the proximal cues within the maze, the spatial location of the target was unchanged with respect to the distal visual room cues. Two trials per day were conducted over 8 training sessions for a total of 16 trials. The latency (s) to reach the target hole, the number of errors, and distance (cm) to reach the target hole were recorded using ImageBM software (see section, “Image analysis”). The day after the last training session, a probe test was conducted without the escape box for 3 min, and the time spent around each hole was recorded to test spatial memory. One month later, a second probe test was performed to assess long-term memory retention.

Fear conditioning test

The contextual and cued fear conditioning test was performed as previously described [28]. Each mouse was placed in a transparent acrylic chamber (26 × 34 × 33 cm) with a grid floor (O’Hara & Co.) and allowed to explore freely for 2 min. A 55-dB white noise was presented for 30 s as a conditioned stimulus (CS), followed by a mild footshock (0.3 mA) during the last 2 s of the CS as an unconditioned stimulus (US). To strengthen the association, 2 more CS-US pairings were presented with a 2-min interstimulus interval to complete the conditioning session. Contextual testing and cued testing were conducted both 1 day and 1 month after the conditioning session. In the contextual testing, each mouse was placed in the same test chamber as used for the conditioning session for 5 min without any stimulus. In the cued testing, each mouse was placed in a triangular chamber (33 × 33 × 33 cm) made of white opaque Plexiglas for 6 min. In the first 3 min, no CS or US was presented, whereas in the last 3 min, the CS was presented. In the conditioning, context, and cued testing, freezing percentage and distance traveled (cm) were calculated using ImageFZ software (see section, “Image analysis”). Freezing was defined as a complete lack of movement of any part of the body for at least 2 s.

T-maze forced alternation test

The T-maze test was performed using an automatic, modified T-maze apparatus (O’Hara & Co.) as previously described [29]. The T-maze comprised white plastic runways with 25-cm high walls partitioned off into 6 areas by sliding doors that open downward automatically. The stem of the T was the S2 area (13 × 24 cm) and the 2 other arms of the T shape were the target arms (A1 and A2, 11 × 20.5 cm each). A1 and A2 connected to the starting area S1 by connecting passages. In the forced alternation task without food, each mouse was subjected to 10 consecutive trials in a session/day for 3 days; each trial comprised a forced-choice run followed by a free-choice run. Each mouse was forced to choose 1 of the target arms of the T (forced-choice run); the door was opened after 10 s and the mouse was returned to the starting area (S1) and allowed to freely choose between both target arms (free-choice run). The percentage of correct trials in which the mice chose the arm opposite their forced-choice run was calculated. The mice were then subjected to the delayed alternation task where 3-, 30-, 60-, or 120-s delays were inserted between the forced- and free-choice runs. Data analysis and acquisition were performed automatically using ImageTM (see section, “Image analysis”).

Image analysis

Behavioral data were collected automatically using the following applications (ImageLD, EP, SI, CSI, BM, FZ, TM, HA, and TS) based on the public domain ImageJ program, and were modified for each test by Tsuyoshi Miyakawa and his colleagues.

Statistical analysis

For the behavioral experiments, statistical analysis was conducted using StatView (SAS Institute, Cary, NC) and data were analyzed using 2-tailed t-tests, paired t-test, one-way analysis of variance (ANOVA), or two-way repeated-measures ANOVA. In the behavioral test battery, we run a series of tests which increases type I error; thus, we consider the results should be corrected for multiple comparisons. For multiple testing in the behavioral test battery, we defined study-wide significance as the statistical significance that survived the false discovery rate (FDR) [30, 31]. Nominal significance was defined as a difference in an index between groups that achieved statistical significance (p < 0.05) but did not survive the FDR correction. All statistical analysis values for the behavioral analyses are included in Additional file 2: Table S2. Statistical analysis for RT-qPCR and Western blotting data was conducted using GraphPad Prism version 8.0.0 (GraphPad Software, Inc., San Diego, CA) and analyzed using Welch’s t or Mann–Whitney tests. Values in graphs and tables are expressed as mean ± SEM.