Animals
T1KO, T1 heterozygotes, and WT littermates on the C57BL/6 background were maintained [7, 9]. Four T1KO or four WT mice were housed per cage, and the mice were group-housed in a room with a 12-h light/dark cycle.
Quantitative biochemical analysis of CS
Purification and quantification of CS in various areas of the brain were performed as described previously [9]. In brief, the mice were sacrificed by decapitation. Various regions of the adult mouse brain were dissected, homogenized, and treated with protease (0.01 mg actinase E, 10 mM CaCl2, 50 mM Tris-HCl, pH 8.0) for 2 days at 55 °C. After trichloroacetic acid treatment, the supernatant was gel filtered (Sephadex G-25, 8.3 ml (PD-10 column), and the flow-through fractions were collected. To analyze the disaccharide composition, fractions were treated with ChABC (5 mIU ChABC in 60 mM CH3COONa, 50 mM Tris-HCl, pH 8.0) for 12 h at 37 °C. The disaccharides were labeled with 2-aminobenzamide (350 mM 2-aminobenzamide, 1 M NaCNBH3 in DMSO/acetic acid (7:3)) for 2 h at 65 °C and analyzed quantitatively using high-performance liquid chromatography (column: YMC pack PA, 4.6 × 250 mm, elution: 16–530 mM NaH2PO4, flow rate: 1 ml/min; detection: 330 nm excitation and 420 nm emission). The disaccharide analysis data are shown in Table 1.
Histochemistry and immunohistochemistry
Histological procedures were carried out according to previously described methods [24]. Antibodies against AGR (rabbit polyclonal) and PV (mouse monoclonal) were purchased from Millipore, and from Swant Inc. (Switzerland), respectively. For tissue preparation, under deep anesthesia with isoflurane, the brain was fixed by cardiac perfusion with PBS followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was dissected out, immersed in the same fixative overnight, and transferred to 20% sucrose in 20 mM PBS, pH 7.4 until it sank. Each brain was frozen in crushed dry ice, and 30 μm-thick consecutive coronal sections were cut on a cryostat and stored at −20 °C until histological staining.
Sections were initially rinsed in 20 mM PBS and incubated in 0.1% Triton X-100 for 15 min at room temperature. After rinsing in 20 mM PBS, the sections were incubated overnight at 4 °C with a mixture of biotinylated-WFA (Sigma-Aldrich) and primary antibodies diluted with 20 mM PBS containing 0.5% skim milk. After rinsing in 20 mM PBS for 15 min, sections were incubated with a mixture of streptavidin-Alexa Fluor® 647 conjugate (1:100; Molecular Probes, Inc., Eugene, OR) and secondary antibodies conjugated to Alexa Fluor®488 or Alexa Fluor®594 (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) for 60 min at 37 °C. After rinsing with 20 mM PBS for 15 min, sections were mounted on MAS®-coated glass slides (Matsunami Glass, Osaka, Japan) and coverslipped with ProLong Gold (Molecular Probes). Sections were observed and digital images were recorded on a confocal laser scanning microscope (FV-1200, Olympus, Tokyo, Japan). TIF files were processed with Photoshop software (Adobe, San Jose, CA). Both brightness and background were adequately adjusted.
For DAB staining, free-floating sections were initially rinsed in 20 mM PBS and incubated in a mixture of 3% hydrogen peroxide and 0.1% Triton X-100 for 15 min at room temperature. 2H6 (Seikagaku Corporation, Tokyo, Japan) was diluted in 20 mM PBS containing 0.5% skim milk. For 2H6 staining, following rinsing in 20 mM PBS for 15 min, sections were incubated with biotinylated anti-mouse IgM secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 30 min at 37 °C. After rinsing with 20 mM PBS for 15 min, sections were incubated in avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories) for 30 min (2H6) or 60 min (WFA) at 37 °C. After rinsing with 20 mM PBS, immunoreaction was visualized in 50 mM Tris-HCl buffer (pH 7.4) containing 0.01% DAB and 0.01% hydrogen peroxide at 37 °C for 5–10 min. Sections were mounted on MAS-coated glass slides (Matsunami Glass), air-dried on a hot plate at 40 °C, and coverslipped with Entellan Neu (Merck, Darmstadt, Germany) after dehydration through ethanol and xylene.
Dilution ratios: were as follows: WFA, 1:50; 2H6, 1:100; anti-PV, 1:500; and anti-AGR, 1:500.
Quantitative morphometry
We initially counted the number of PV (+) cells. However, that method was not suitable for exact quantification of PV (+) cells because the staining of PV (+) cells varied widely. Thus, we switched to the following quantification methods. The fluorescent intensity of the immunohistochemical and WFA histochemical images was statistically calculated using Image J. Double- (AGR and WFA) or triple (AGR, WFA, and PV)-labeled confocal images were used for the intensity analysis. AGR+ pixels were extracted by masking with an intensity threshold (>50). The average intensity of the AGR+ pixels was calculated for each AGR, WFA, or PV image. The average intensity data were grouped by genotype and brain area. Two-way factorial ANOVA with Bonferroni post-hoc tests were performed for statistical analysis.
Animal behavioral tests
A battery of mouse behavioral tests was done as described previously [25, 26]. All behavioral tests were carried out with male mice. A general health check and neurological screen were conducted as previously described [27]. The order in which mice were subjected to tests was counterbalanced. Raw data were disclosed in the Mouse Phenotype Database (http://www.mouse-phenotype.org/). T1KO and WT mice (n = 20 each) at 8 weeks of age were tested. The tests that showed statistically significant differences in T1KO vs. WT were:
(I) Open field test: This test was performed to evaluate locomotor activity and emotional response [21]. The apparatus was a transparent square cage (42 × 42 × 30 cm; Accuscan Instruments, Columbus, OH). The center of the floor was illuminated at 100 lx. Each mouse was placed in the open field apparatus and recorded for 120 min. Total distance traveled (cm), vertical activity (rearing measured by counting the number of photobeam interruptions), time spent in the center area (20 × 20 cm), and the beam-break counts for stereotyped behaviors were measured.
(II) Social interaction test in a novel environment [27]: Two mice of the same genotype that were previously housed in different cages were placed in a box together (40 × 40 × 30 cm; O’Hara & Co., Tokyo, Japan) and allowed to explore freely for 10 min. Mouse behavior was analyzed automatically using ImageSI software. The total duration of contacts (s), number of contacts, total duration of active contacts (s), mean duration per contact (s), and total distance traveled (cm) were measured. Active contact was defined as follows: images were captured at three frames per second, and distance traveled between two successive frames was calculated for each mouse. If the two mice contacted each other and the distance traveled by either mouse was 5 cm or more, the behavior was considered an “active contact.”
(III) Three-chambered social approach test: The test for sociability and social novelty preference was conducted as previously described [28, 29]. For habituation to the test environment, stranger mice were placed in a small cylindrical cage with vertical bars that was put in a corner of the chamber prior to the test. Test mice were placed in the middle chamber and allowed to explore and habituate to the chambers for 10 min just before the first session. In the first session, an unfamiliar mouse (stranger 1) was put in the cage and placed in a corner of the left or right side chamber. The selection of the left or right side chamber was counterbalanced across test mice. An empty cage was placed in the corner of the other chamber. Then the test mouse was placed in the middle chamber and allowed to explore the three chambers for 10 min. In the second session, another unfamiliar mouse (stranger 2) was placed in the cage that had been empty during the first session. Then the test mouse was placed in the middle chamber and allowed to explore for 10 min. Total distance travelled, average locomotion speed, and the amount of time spent around the cages were measured. Data acquisition and analysis were performed automatically using ImageSI.
(IV) Startle response/prepulse inhibition test: A startle reflex measurement system (O’Hara & Co.) was used to measure startle response to a loud noise and prepulse inhibition of the startle response. A test session began by placing a mouse in a plastic cylinder where it was left undisturbed for 10 min. White noise (40 ms) was used as the startle stimulus for all trial types. The startle response was recorded for 400 ms starting with the onset of the startle stimulus. The background noise level was 70 dB. The peak startle amplitude was the dependent variable. A test session consisted of six trial types (e.g., two types of startle-stimulus-only trials, and four types for prepulse inhibition trials). The intensity of the startle stimulus was either 110 or 120 dB. The prepulse sound was presented 100 ms before the onset of the startle stimulus, and its intensity was 74 or 78 dB (20 ms). Four combinations of prepulse and startle stimuli were used (74–110, 78–110, 74–120, and 78–120 dB). Six blocks of the six 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). Behavioral tests that were not significantly different between genotypes are listed in Table 2. Behavioral data were obtained automatically by applications (ImageLD, EP, SI, CSI, PS, FZ, TS, HCSI, and BM) based on the public domain Image J program (http://rsb.info.nih.gov/ij/) and modified for each test by the authors. Statistical analysis was conducted using StatView (SAS Institute, Cary, NC). Data were analyzed with the Student’s t-test, paired t-test, one-way ANOVA, or two-way repeated measures ANOVA. Values in graphs are presented as the mean ± SEM.