Biochemical and histochemical characteristics of T1KO

T1KO mice were fertile and showed no macroscopic abnormalities in the brain. However, various regions of the brain had only 50% of the amount of CS compared to WT, including significant differences in the cortex and diencephalon (Fig. 1).

Next, we examined whether microscopic abnormalities were found in T1KO brain. Because high levels of CS should be detected in PNNs of WT, we histochemically examined WFA-labeled PNNs using diaminobenzidine (DAB) staining and fluorescent studies. DAB staining of WFA, which represents PNNs, showed that PNNs were largely reduced in the cingulate cortex of T1KO (Fig. 2a, b, e-f), and to some extent in the somatosensory cortex of T1KO (Fig. 2d and h). Using 2H6, an antibody against CS that also tends to label extracellular matrix other than PNNs, we confirmed that CS staining in such areas was much reduced (Fig. 2c and g).

We quantitatively analyzed WFA, anti-AGR, and anti-PV immunofluorescence by assessing the intensity (Fig. 2i-p) following triple fluorescent staining. Somatosensory cortex showed a reduction in WFA (+) PNNs (Fig. 2i and m), but the intensities of anti-AGR and anti-PV immunofluorescence, which is thought to represent the amounts of AGR in PNNs and PV (+) cells, respectively, were not different in T1KO (Fig. 2m). We observed similar patterns in the cingulate cortex (Fig. 2j and n), hippocampus (Fig. 2k and o), and thalamic reticular nucleus of the T1KO (Fig. 2l and p). These results suggest that T1KO have reduced CS in PNNs but the core proteins remain, independent of CS.

Quantitative analysis of these fluorescent results (Fig. 2i-p) confirmed this observation (Fig. 3). In all examined areas in T1KO, the intensities of anti-AGR and anti-PV were not changed, but the intensity ratios of WFA (+) PNNs per AGR (+) area were selectively decreased (Fig. 3). We also confirmed that AGR immunoreactivity was not changed between brains of WT and T1KO using immunoblotting (Additional file 1 Figure S1).

Behavioral abnormalities in T1KO

We performed a battery of more than 20 behavioral tests and found the following abnormalities in T1KO compared to WT: general health (body weight), open field test, rota-rod test, acoustic startle responses, general activity, and social preference (stay time) (Fig. 4). The total distance traveled in the open field test, which measures voluntary activity in a novel environment, was significantly higher in T1KO than WT, suggesting that T1KO showed hyperlocomotive activity. Acoustic startle tests, which measure reflex movement when a sudden loud sound stimulus is given, revealed that T1KO had much larger responses than WT. In contrast, other tests including learning and memory were not impaired (Table 2). These results suggest that T1KO show several abnormal behaviors that involve higher brain functions, probably due to the reduced CS concentration in PNNs.

Test	Controls		Mutants		F value	p value
	Mean	SEM	Mean	SEM
General health/neurological screen
body temperature (°C)	36.85	0.141	37.28	0.164	2.747	0.1049
grip strength (n)	0.554	0.02	0.577	0.012	1.038	0.3141
wire hang (latency to fall, s)	39.512	5.428	29.507	2.879	3.204	0.0808
Light/dark transition
latency to light (s)	44.143	5.829	56.1	6.621	1.293	0.2619
light distance (cm)	622.129	46.667	609.563	41.119	0.034	0.8548
dark distance (cm)	1555.886	65.493	1628.983	35.355	1.147	0.2904
number of transitions	32.714	2.23	28.967	1.965	1.32	0.2571
stay time in light (s)	158.071	9.869	147.5	10.065	0.423	0.5189
Open field
stereotypic counts	6911.643	520.353	7369.367	526.983	0.289	0.5939
vertical activity	348.357	53.683	275.4	43.994	0.966	0.3312
center time (s)	633.986	121.934	813.077	111.923	0.945	0.3365
Elevated plus maze
number of total entry (times)	36.071	2.319	36.833	2.229	0.044	0.835
entry into open arms (%)	13.393	2.567	13.863	1.572	0.027	0.8715
total distance travelled (cm)	1806.893	92.545	1880.087	73.559	0.343	0.5614
time on open arms (times)	3.443	1.125	2.923	0.744	0.152	0.6985
Hot plate
latency (s)	6.403	0.386	6.318	0.394	0.018	0.8944
Social interaction (novel environment)
duration of contacts (s)	68.7	9.138	88.707	7.29	2.605	0.1222
number or contacts (times)	40.143	3.845	45.333	2.462	1.358	0.2577
mean duration of contacts (s)	1.786	0.263	1.993	0.187	0.4	0.5341
distance travelled (cm)	2957.586	285.871	3196.793	153.133	0.654	0.4283
Porsolt forced swim
day1 immobility (%)	41.8131	3.617	46.252	2.314	1.099	0.3009
day2 immobility (%)	57.747	3.627	54.268	2.624	0.569	0.4551
Gait analysis
front swing/stride duration	41	0.508	40.969	0.43	0.002	0.9662
front brake/stride duration	28.115	0.909	27.234	0.715	0.512	0.4784
front propel/stride duration	30.896	0.871	31.795	0.645	0.635	0.4302
front stance width	1.946	0.062	1.921	0.038	0.134	0.7164
front step angle	62.308	1.762	60.066	1.075	1.269	0.2666
front paw angle	−0.235	0.866	0.89	0.455	1.591	0.2144
hind brake/stride duration	14.142	0.831	15.564	0.364	3.373	0.0737
hind step angle	53.746	1.757	55.321	1.627	0.338	0.564
hind paw angle	−0.788	0.953	−0.983	0.519	0.038	0.8471
Barnes maze
time spent around target (probe test, 1 day)	40.538	4.748	39.143	4.067	0.042	0.8385
time spent around target (probe test, 1 month)	32.154	5.78	30.172	4.224	0.072	0.7904
time spent around target (reversal test, 1 day)	31.077	4.118	28.214	3.227	0.27	0.6064
Cued and contextual fear conditioning
conditioning (freezing, %)	57.756	2.66	56.821	1.83	0.82	0.7764
context test, 1 day (freezing, %)	70.082	3.444	66.333	3.372	0.453	0.5053
cued test, with tone,1 day (freezing, %)	89.158	3.641	88.147	2.004	0.069	0.7945
context test, 30 days (freezing, %)	79.412	5.225	79.481	3.222	0.0001376	0.9907
cued test, pretone period, 30 days (freezing, %)	60.603	4.475	49.672	4.306	2.345	0.1342
cued test, with tone, 30 days (freezing, %)	90.183	3.079	87.86	1.944	0.425	0.5186
Tail suspension
immobility (%)	37.255	3.747	41.75	2.603	0.957	0.334
24 h home cage monitoring
activity light period (A.U.)	478,882.065	30,477.038	670,800.611	107,540.908	2.948	0.1167
activity dark period (A.U.)	1,033,998.833	66,355.759	1,385,925	191,066.128	3.027	0.1125
number of particle dark period	1.175	0.02	1.196	0.022	0.482	0.5034
	Stranger		Empty or Familiar		t value	p value (paired-t)
	Mean	SEM	Mean	SEM
Social approach test (Stranger vs Empty)
number of entry around cage (controls)	9	1.043	7.714	0.952	1.178	0.26
time spent around cage (mutants)	209.241	12.352	137.448	10.928	0.627	0.5359
Social novelty preference test (Stranger vs Familiar)
number of entry around cage (controls)	8.357	0.887	8.786	1.11	−0.405	0.6918
number of entry around cage (mutants)	9.862	0.851	10.034	0.863	−0.16	0.8742
time spent around cage (controls)	165.071	16.433	148.5	28.882	0.435	0.6708
time spent around cage (mutants)	175.31	17.16	156.379	13.814	0.673	0.5063

The tests that were significantly different between T1KO and WT are displayed in Fig. 4. The results of the tests that were not significantly different are listed here. Each group was composed of 20 male mice at the age of 12 weeks

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.