Abstract

Down syndrome (DS) is the most common cause of mental retardation. Although structural and neurogenic abnormalities have been shown in the brains of DS patients, the molecular etiology is still unknown. To define it, we have performed structural and histological examinations of the brains of Ts1Cje and Ts2Cje, 2 mouse models for DS. These mice carry different length of trisomic segments of mouse chromosome 16 that are orthologous to human chromosome 21. At 3 months of age, ventricular enlargements were observed in both Ts1Cje and Ts2Cje brains at a similar degree. Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles (LVs) and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared with diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the genes that are responsible for these abnormal phenotypes and could be relevant to the mental retardation associated with DS.

Introduction

Down syndrome (DS) or trisomy 21, with an incidence of 1 in 700 live birth, is the most common autosomal aneuploidy and genetic cause of mental retardation (Epstein 2001). Because most of human chromosome 21 is orthologous to distal end of mouse chromosome 16, several mouse models of DS with full or segmental trisomy for mouse chromosome 16 have been established. Ts(1716)65Dn (hereafter called Ts65Dn) (Davisson et al. 1990) and Ts(1216C-tel)1Cje (Ts1Cje) mice (Sago et al. 1998) carry trisomic segments of mouse chromosome 16 that contain regions orthologous to human chromosome 21. Additionally, the Ts[Rb(12.1716)]2Cje (Ts2Cje) mouse has been recently established after a fortuitous translocation of the Ts65Dn marker chromosome to chromosome 12 (Villar et al. 2005). Both Ts65Dn and Ts2Cje carry a trisomic segment extending from the gene encoding mitochondrial ribosomal protein L39 (mrpl39) to the zinc finger protein 295 (znf295) gene. In contrast, the Ts1Cje mouse has a smaller extra segment extending from Cu/Zn-superoxide dismutase (sod1) to znf295, but sod1 is functionally excluded because of a mutation in the gene (Fig. 1) (Sago et al. 1998). Many groups, including our own, have performed gene expression studies in Ts65Dn and Ts1Cje mice and have shown a near 1.5-fold mean overexpression of the triplicated genes (Amano et al. 2004; Kahlem et al. 2004; Dauphinot et al. 2005). Both Ts65Dn and Ts1Cje mice show DS-related phenotypes that include cognitive and behavioral impairments (Reeves et al. 1995; Sago et al. 1998), craniofacial abnormalities (Richtsmeier et al. 2000, 2002), increased oxidative stress (Shukkur et al. 2006; Lockrow et al. 2009), and abnormal dendritic spine morphology (Belichenko et al. 2004, 2007). It has also been shown that long-term potentiation is greatly decreased in the dentate gyrus (DG) of both Ts1Cje and Ts65Dn mice (Kleschevnikov et al. 2004; Belichenko et al. 2007).

Figure 1.

Trisomic segments in Ts1Cje and Ts2Cje DS model mice. Most part of the long arm of human chromosome 21 is orthologous to distal end of mouse chromosome 16. Information including genomic size and number of genes in the trisomic mouse chromosome 16 partial segments and human chromosome 21 is based on NCBI databases, mouse build 37.1 (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) and build 36.3 of human genome (http://www.ncbi.nlm.nih.gov/mapview/). Note that the sod1 gene in the trisomic segment of Ts1Cje is inactivated by insertion of NEO cassette (Sago et al. 1998). *, number of protein-coding genes on human chromosome 21; **, number of protein-coding genes conserved between human chromosome 21 and mouse chromosome 16.

Figure 1.

Trisomic segments in Ts1Cje and Ts2Cje DS model mice. Most part of the long arm of human chromosome 21 is orthologous to distal end of mouse chromosome 16. Information including genomic size and number of genes in the trisomic mouse chromosome 16 partial segments and human chromosome 21 is based on NCBI databases, mouse build 37.1 (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) and build 36.3 of human genome (http://www.ncbi.nlm.nih.gov/mapview/). Note that the sod1 gene in the trisomic segment of Ts1Cje is inactivated by insertion of NEO cassette (Sago et al. 1998). *, number of protein-coding genes on human chromosome 21; **, number of protein-coding genes conserved between human chromosome 21 and mouse chromosome 16.

Several studies have shown that the brains of persons with DS are smaller than normal brain, with disproportionately smaller cerebellar, brainstem, frontal lobe, and hippocampal volumes (Weis et al. 1991; Kesslak et al. 1994; Raz et al. 1995; Aylward et al. 1999). Additionally, a study demonstrated increases of ventricular volumes in the DS adults (Pearlson et al. 1998). White et al. (2003) showed that cerebral spinal fluid was increased in adults with DS without dementia, suggesting that there was ventricular enlargement in DS adults. Furthermore, enlargement of the third ventricles has recently been reported in newborn infants with DS (Schimmel et al. 2006). Thus, enlargement of ventricles in individuals with DS may be important in the etiology of the mental retardation in DS, but so far there are no reports demonstrating ventricular enlargement in mouse models for DS.

In addition to the morphological changes, neurological abnormalities in DS are also characterized by reduced number of cortical neurons, malformed dendritic trees and spines, impaired lamination of cortex, and abnormal synapses (Takashima et al. 1981; Wisniewski et al. 1984; Golden and Hyman 1994). Furthermore, in vitro experiments using neurospheres derived from the cortices of 8–18 weeks DS fetuses have demonstrated a decreased ability to differentiate into neurons, suggesting that neurogenesis is disturbed in the brain with DS (Bahn et al. 2002). Additionally, impaired cell proliferation in 2 neurogenic regions, the DG and the germinal matrix of the LV, has been described in postmortem brain from DS fetuses (Contestabile et al. 2007). These reports allow us to hypothesize that the hypoplasia of the adult DS brain may be related to developmental defects in the processes of neurogenesis. In addition to the findings from DS brains, it has been shown that embryonic and adult neurogenesis is also impaired in the neocortex and hippocampus of Ts65Dn mouse model, respectively (Clark et al. 2006; Chakrabarti et al. 2007), but to date no such study is available for Ts1Cje.

In the present study, we examined the ventricular size of Ts1Cje and Ts2Cje brains using histological and magnetic resonance imaging (MRI) analyses and found enlargement in both strains of mice. Furthermore, we also demonstrated that neurogenesis was decreased in both DS mouse models at both prenatal and postnatal stages.

Materials and Methods

Animal Maintenance and Genotyping

Ts1Cje mice were maintained by crossing carrier males with C57BL/6J (B6) females. A genotyping of Ts1Cje mouse was performed by polymerase chain reaction (PCR) using multiplex primers for neomycin resistance gene (Neo) and glutamate receptor, ionotropic, kainate 1 (internal control) as previously described (Amano et al. 2004). We also maintained Ts[Rb(12.1716)]2Cje (Ts2Cje) (Villar et al. 2005) and Ts1Cje on C57BL/6JEi and C3H/HeSnJ hybrid (B6/C3H) background. Ts2Cje founder female mice were mated to B6/C3H F1 male mice to establish a breeding colony. Cultured tail fibroblasts prepared at 6 days of age were used for karyotype analysis of metaphase spreads to determine the genotype by fluorescent in situ hybridization using a BAC clone carrying the mouse DS cell adhesion molecule (dscam) genomic region (mapping to the distal part of mouse chromosome 16) as a probe. Furthermore, the genotype was confirmed by quantitative PCR as previously described (Villar et al. 2005). To generate Ts1Cje mice on the B6/C3H hybrid background, Ts1Cje mice on the B6 background were crossed with C3H/HeSnJ mice and the resulting Ts1Cje mice were crossed to B6/C3H F1 mice. All mice were housed less than 5 per cage with a 12-hr light–dark cycle and ad libitum access to food and water. All experimental procedures were performed in accordance with the guidelines of the Animal Experiments Committee of RIKEN Brain Science Institute. For the experiments of 12-day-old, 1-month-old, 3-month-old, and 6-month-old mice, we used male mice exclusively.

Nissl Staining

Mice were deeply anesthetized with pentobarbital (0.3 mg/g body weight, intraperitoneal administration [i.p.]) and perfused transcardially with saline and 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) using a 24-gauge needle on a handheld syringe at rate of less than 0.5 mL/min. Brains were postfixed in the same fixation solution at 4 °C for 2 days, submerged in 30% sucrose/PBS until saturated, and then frozen in Tissue-Tek OCT (Miles, Elkhart, IN) for cutting 30-μm sections using a cryostat (CM1900, Leica, Wetzlar, Germany). The brain sections were stained in 0.25% thionin solution, dehydrated, and coverslipped in MGK-S (Matsunami Glass, Osaka, Japan) and observed at low magnification with a light microscope (AX80, Olympus, Tokyo, Japan).

Measurement of Ventricular Sizes in the Brain Sections

Equivalent sections of Ts1Cje mouse and wild-type littermate brains were chosen on the basis of common morphological landmarks (Paxinos and Franklin 2001). Images of stained tissues were converted into TIFF format using Adobe Photoshop Elements 2.0. The areas of the brain substance and ventricles were measured using NIH ImageJ 1.39u (developed at US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij/). Two images from each individual mouse were measured. Student's t-test was used to assess statistical significance between groups.

MRI Study

All MRI was performed with a vertical-bore 9.4-T Bruker AVANCE 400WB imaging spectrometer with a 250 mT/m actively shielded imaging gradient insert (Bruker BioSpin, Ettlingen, Germany). For in vivo MRI scanning, mice were anesthetized with 1.5–2% isoflurane and secured using a head holder with bite bar to reduce motion artifacts. Mice were subjected to a T2-weighed MRI study. Images were obtained using a 2-dimensional multislice spin echo sequence with the following parameters: field of vision = 30 mm, acquisition matrix = 256 × 512, slice thickness = 0.5 mm (for coronal and sagittal), 0.3 mm (for horizontal), time of repetition/time of echo = 7773.1/55.6 ms, and number of averages = 4. Thirty-one coronal, 22 sagittal, and 23 horizontal slices were acquired, covering the entire brain. The ventricular areas of all coronal MRI images from each mouse were quantified using the NIH ImageJ program. The area of olfactory bulb was also measured from horizontal MRI images. The volumes of ventricles, olfactory bulbs, and total brain tissue were calculated by following formula: volume (mm3) = each area (mm2) × (slice thickness + gap among images).

In Vivo BrdU Labeling

For detection of adult neurogenesis, mice were injected intraperitoneally for 8 consecutive days with 5-bromo-2′-deoxyuridine (BrdU; 100 mg/kg body weight in PBS, Sigma-Aldrich, St Louis, MO). Twenty-four hours following the last BrdU injection, the mice were anesthetized with Avertin (250 mg/kg of tribromoethanol, i.p.) and transcardially perfused with cold saline followed by 4% PFA/PBS using a 24-gauge needle on a handheld syringe at rate of less than 0.5 mL/min. Brains were collected and postfixed for 2 days in 4% PFA at 4 °C, then cryoprotected in 30% sucrose at 4 °C, frozen in 50% Tissue-Tec in 15% sucrose, and kept at –80 °C until processed for immunohistochemistry. For the examination of prenatal neurogenesis, pregnant Ts1Cje and Ts2Cje females at embryonic day 13.5 were administrated with 1 pulse of 50 mg/kg BrdU and the embryos were killed at 24 h later. The heads were rotated in 4% PFA/saline for 3 days at 4 °C, then cryoprotected in 30% sucrose at 4°C, frozen in 50% Tissue-Tec in 15% sucrose, and kept at –80 °C until processed for immunohistochemistry.

Immunohistochemistry for Detection of Doublecortin- and BrdU-Positive Cells on Adult Brain Sections

Brains were sectioned using a cryostat (30 μm) and mounted onto Superfrost MS-coated slides. Equivalent sections of DS mouse and respective wild-type littermate brains stained with thionin were chosen based on common morphological landmarks (Paxinos and Franklin 2001). Slices were incubated in 0.3% hydrogen peroxide for 10 min at room temperature, rinsed PBS, and then incubated in 90 °C preheated Retrievagen (BD Biosciences, Boston, MA) for 10 min. After cooling to room temperature, slides were washed with PBS and incubated with mouse on mouse blocking regent (Vector Laboratories, Burlingame, CA) for 1 h. After washing with PBS, slides were blocked with blocking solution (0.3% Triton X-100 and 10% horse serum in PBS) containing Avidin block solution (Vector Laboratories) for 1 h. Sections were then incubated overnight at 4 °C with anti-doublecortin (DCX) C-18 polyclonal antibody (1:100 dilution in 1% normal horse serum; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-BrdU antibody (1:200, BD Biosciences) containing Biotin blocking solution (Vector Laboratories). After washing with PBS, brain sections were processed for 1 h at room temperature in biotinylated horse anti-goat (for DCX) or anti-mouse IgG antibody (for BrdU) (1:200; Vector Laboratories). Labeled cells were visualized using the ABC system (Vectastain Elite; Vector Laboratories) and metal-enhanced chromogen diaminobenzidine tetrachloride (DAB; Pierce, Rockford, IL). Sections were then counterstained with hematoxylin, dehydrated, and coverslipped in MGK-S (Matsunami Glass). Photomicrographs were acquired by using light microscopes, AX80 (Olympus) and BZ-8000 (Keyence, Osaka, Japan). For the quantification of the immunopositive cell number, a series of Z-stack images were acquired (5 μm thickness) on BZ-8000, and cells were counted according to unbiased stereological 3-dimensional cell-counting methods (Williams and Rakic 1988). We counted DCX- and BrdU-positive cells in 12 DGs and 7 LVs of each genotype (n = 3 in each genotype) in a blinded manner.

Immunohistochemistry for Detection of BrdU- and Ki67-Positive Cells on the Embryonic Brain Sections

For detection of BrdU-positive cells with DAB, frozen coronal sections from embryonic brain (24 h after 1 pulse with BrdU, E14.5, 30 μm thickness) were immunostained with BrdU antibody as described above, counterstained with hematoxylin, dehydrated, and coverslipped in MGK-S (Matsunami Glass). For simultaneous detection of Ki67 and BrdU by immunofluorescence, brain sections were incubated overnight at 4 °C with rabbit anti-Ki67 polyclonal (1:500; Novacastra, Norwell, MA) and mouse anti-BrdU monoclonal antibodies (1:200; BD Biosciences) after antigen retrieval and blocking process. After washing with PBS, brain sections were then incubated for 1 h at room temperature with Alexia 488-conjugated donkey anti-rabbit IgG (1:400; Invitrogen, Carlsbad, CA) and Alexia 594-conjugated donkey anti-mouse IgG (1:400; Invitrogen). The nuclei were then stained with 4′,6-diamino-2-phenylindole (DAPI), and the brain sections were coverslipped in Prolong Gold antifade reagent (Invitrogen). Photomicrographs were acquired by using a light microscope AX80 (Olympus) and fluorescence microscope BZ-8000 (Keyence). In the detection with DAB, BrdU-positive and -negative cells in embryonic cortex (100 μm wide) were counted in a blinded manner (n = 3 for Ts2Cje and n = 4 for Ts1Cje). In addition, we also measured the area of BrdU-positive cells by ImageJ software. The ratio of BrdU-positive cells or area in the ventricular zone (VZ)/subventricular zone (SVZ), the intermediate zone (IZ), and the subplate–cortical plate (SP/CP) were calculated. SP/CP refers to the area bounded by the large, pale SP cells and the border between the CP and the marginal layer. The area between the SVZ and SP is referred to as the IZ. In the double-labeling immunofluorescence for Ki67 and BrdU, BrdU(+), and BrdU(+)/Ki67(−) cells and DAPI-stained nuclei were counted in 3 counting boxes (50 × 50 μm frame in 5-μm thick Z-stack images [0.5 μm steps]) in each slice by stereological method on BZ-8000 fluorescent microscope (n = 2 in each genotype).

Results

Enlarged Brain Ventricles in Ts1Cje Mice

Ts1Cje mice were maintained on C57BL/6J (B6) background. We stained the coronal brain sections of Ts1Cje mice and wild-type littermates at postnatal days 12 and months 1, 3, and 6 with Nissl dye thionin (Fig. 2A and Supplementary Fig. S1A), and the areas of the ventricles were quantified by the NIH ImageJ program (Supplementary Fig. S1B). At all ages examined, the lateral and/or third ventricles were enlarged in the brains of Ts1Cje mice compared with those of wild-type littermates, although the degrees of enlargement varied among individual mice. Next, we performed MRI scans to confirm the ventricular enlargement in Ts1Cje mice (Fig. 2B–E). Coronal, sagittal, and horizontal images of T2-weighted MRIs were captured to identify the cerebrospinal fluid (CSF)-containing ventricles, which were detected as white areas (Fig. 2C and Supplementary Fig. S2A). As expected, the ventricles, especially the LVs, were dramatically expanded in Ts1Cje mice at all ages examined (Fig. 2C and Supplementary Fig. S2A). Analyses of all serial coronal MRI slices with image analyzing software revealed that the lateral ventricular volumes of Ts1Cje mice are approximately twice as large as those of littermate controls at all examined ages, but the differences in the volumes of third and fourth ventricles were not statistically significant (Fig. 2D). No obstructions of the fourth ventricle or of the aqueduct were observed in Ts1Cje mice on histological and MRI analyses (Supplementary Fig. S2B; data not shown). In contrast to the differences in the ventricular size, no statistical difference between Ts1Cje and wild-type littermates was detected for whole brain volumes excluding ventricles and brain stem (Fig. 2E). Similarly, the mean wet weights of the brains were nearly the same in the 2 strains at 3 months of age (mean [g] ± standard error, wild type, 0.467 ± 0.004; Ts1Cje, 0.466 ± 0.007; n = 5), although the body weights of Ts1Cje mice were lighter than those of wild-type littermates (Supplementary Fig. S3A). Taken together, these results indicate that brain ventricles are enlarged in Ts1Cje mouse without significant volumetric change of entire brain parenchyma.

Figure 2.

Enlarged brain ventricles in Ts1Cje. (A) Nissl staining of coronal brain sections (at bregma −1.2 mm for P12, -1.4 mm for 1 M, −1.5 mm for 3 and 6 M) from Ts1Cje (Ts1) and wild-type (WT) littermates on B6 background at 12 days and 1, 3, and 6 months of age. Prominent enlargements of ventricles are observed in Ts1Cje. Scale bar: 1 mm. (B) Schematic representation of coronal sectioning on brain MRI. Thirty-one cortical images were acquired with the 0.5-mm gap between the images. The 12th and 17th images from the most anterior part of nose indicated by arrows were used as slices a and b in C. (C) Typical T2-weighted MRI images (slices a and b) from WT littermates (upper panels) and Ts1 (lower panels) on B6 background at 1, 3, and 6 months of age. Note that the LVs were enlarged in Ts1 mice (white areas). (D) Volume of each brain ventricle was calculated by summing-up the area of each section multiplied by slice thickness. The value is the ratio of each ventricle volume to whole brain volume (mean ± standard error of the mean; n = 3 in each group and each age). LV is volumetrically increased in Ts1 at all examined ages. (E) The volumes of whole brain parenchyma excluding brain stem and brain ventricles. No significant change in whole brain volume is detected between 2 genotypes at all examined ages. Statistical significance was determined with the Student's t-test. *P < 0.05, significantly different from WT. D3V, dorsal third ventricle; V3V, ventral third ventricle; and 4V, fourth ventricle.

Figure 2.

Enlarged brain ventricles in Ts1Cje. (A) Nissl staining of coronal brain sections (at bregma −1.2 mm for P12, -1.4 mm for 1 M, −1.5 mm for 3 and 6 M) from Ts1Cje (Ts1) and wild-type (WT) littermates on B6 background at 12 days and 1, 3, and 6 months of age. Prominent enlargements of ventricles are observed in Ts1Cje. Scale bar: 1 mm. (B) Schematic representation of coronal sectioning on brain MRI. Thirty-one cortical images were acquired with the 0.5-mm gap between the images. The 12th and 17th images from the most anterior part of nose indicated by arrows were used as slices a and b in C. (C) Typical T2-weighted MRI images (slices a and b) from WT littermates (upper panels) and Ts1 (lower panels) on B6 background at 1, 3, and 6 months of age. Note that the LVs were enlarged in Ts1 mice (white areas). (D) Volume of each brain ventricle was calculated by summing-up the area of each section multiplied by slice thickness. The value is the ratio of each ventricle volume to whole brain volume (mean ± standard error of the mean; n = 3 in each group and each age). LV is volumetrically increased in Ts1 at all examined ages. (E) The volumes of whole brain parenchyma excluding brain stem and brain ventricles. No significant change in whole brain volume is detected between 2 genotypes at all examined ages. Statistical significance was determined with the Student's t-test. *P < 0.05, significantly different from WT. D3V, dorsal third ventricle; V3V, ventral third ventricle; and 4V, fourth ventricle.

Enlarged Brain Ventricles in Ts2Cje Mice

Next, we examined the ventricles of the Ts2Cje mouse, which carries the Ts65Dn trisomic segment, which is larger than that of Ts1Cje mouse (Fig. 1). Because repeated attempts to place the Ts65Dn chromosome on inbred backgrounds have failed because of drastically reduced litter size and failure to recover trisomic mice (JAX Notes 2005), Ts2Cje mice were maintained on the hybrid background of C57BL/6JEi and C3H/HeSnJ (B6/C3H). Similar to Ts1Cje, the mean volume of the LVs in Ts2Cje was approximately twice that of wild-type littermates at 3 months of age on MRI analyses (Fig. 3A, B). In addition, both the dorsal and ventral third ventricles were also significantly enlarged in Ts2Cje. For exact comparison, we further analyzed the brain of Ts1Cje on the B6/C3H hybrid background that was identical to that of Ts2Cje (Fig. 3D, E). Ts1Cje mice again showed the enlargement of lateral and dorsal third ventricles compared with wild-type littermates, and the degrees of enlargements are mostly similar in both B6-inbred and B6/C3H hybrid background, suggesting that the genetic background of these strains did not much affect this phenotype. Although the LVs in Ts2Cje seemed to be smaller than those in Ts1Cje on this hybrid background, this difference was not statistically significant (Ts1Cje vs Ts2Cje, t4 = 2.35, P = 0.065; Student's t-test). By contrast, whole brain volumes of Ts1Cje and Ts2Cje were almost same as those of the respective wild-type littermates (Fig. 3C, F), although the body weights of both DS models were lighter than those of their respective wild-type littermates (Supplementary Fig. S3B). No obstructions of fourth ventricle or aqueduct were observed in both DS models on the B6/C3H hybrid background (data not shown). Thus, in both DS models, we found ventricles enlarged to a similar degree, suggesting that the triplicated segment of Ts1Cje contains the genes responsible for the enlargement of brain ventricles in both Ts1Cje and Ts2Cje.

Figure 3.

Enlarged brain ventricles in Ts2Cje. MRI analyses was performed on the brains from Ts2Cje (Ts2) (AC) and Ts1Cje mice (Ts1) (DF) on B6/C3H hybrid background at 3 months of age. (A, D) Coronal T2-weighted MRI images (slices a and b) of the brain from Ts2 (A), Ts1 (D), and respective wild-type (WT) littermates. Expanded LV and third ventricle (3V) were observed in both Ts1 and Ts2 mice. (B, E) The ratios of each ventricular volume to whole brain volume indicate expansion of ventricles in both DS models (mean ± standard error of the mean; n = 3 in each group and each age). (C, F) No significant change in whole brain volumes is detected in both mice Ts1 and Ts2 compared with respective wild-type littermates. Statistical significance was determined with the Student's t-test. ***P < 0.001, **P < 0.01, *P < 0.05, significantly different from WT.

Figure 3.

Enlarged brain ventricles in Ts2Cje. MRI analyses was performed on the brains from Ts2Cje (Ts2) (AC) and Ts1Cje mice (Ts1) (DF) on B6/C3H hybrid background at 3 months of age. (A, D) Coronal T2-weighted MRI images (slices a and b) of the brain from Ts2 (A), Ts1 (D), and respective wild-type (WT) littermates. Expanded LV and third ventricle (3V) were observed in both Ts1 and Ts2 mice. (B, E) The ratios of each ventricular volume to whole brain volume indicate expansion of ventricles in both DS models (mean ± standard error of the mean; n = 3 in each group and each age). (C, F) No significant change in whole brain volumes is detected in both mice Ts1 and Ts2 compared with respective wild-type littermates. Statistical significance was determined with the Student's t-test. ***P < 0.001, **P < 0.01, *P < 0.05, significantly different from WT.

Short Anteroposterior Length of the Brain in Ts1Cje and Ts2Cje

Consistent with the previous report of Ts1Cje's brachycephalic skull (Richtsmeier et al. 2002), our analyses on sagittal MRI sections revealed that the anteroposterior length of the Ts1Cje brain on the B6 background was short, although the difference did not reach statistical significance (Supplementary Fig. S4A). However, the lengths of the brains of both Ts1Cje and Ts2Cje mice on the B6/C3H hybrid background were significantly decreased (Supplementary Fig. S4B).

Impaired Adult Neurogenesis in Ts1Cje and Ts2Cje Mouse Brains

Next, we investigated neurogenesis in the SVZ by immunohistochemistry of DCX, a neuroblast marker protein (Brown et al. 2003), in Ts1Cje on the B6/C3H hybrid background at 3 months of age. Reduced numbers of DCX-positive cells was detected in the SVZ of Ts1Cje compared with wild-type littermates (Fig. 4A–C). We also found that the number of DCX-positive cells in hippocampal DG, which is another neurogenic region in adult rodents, was decreased in Ts1Cje (Fig. 4D, E). The counting of DCX-positive cells in a blinded manner revealed that the number of neuroblasts was reduced by approximately 30% and 50% in the SVZ and DG (granule cell layer [GCL] and subgranular layer [SGL]), respectively, of adult Ts1Cje mice compared with wild-type littermates (Fig. 4F). Similar levels of reductions of DCX-positive cells in the 2 neurogenic regions were also observed in Ts2Cje (Fig. 5).

Figure 4.

Decreased DCX-positive neuroblasts in the SVZ and DG of Ts1Cje. Immunohistochemistry using anti- DCX antibody was performed on the brain sections from Ts1Cje (Ts1) mice on the B6/C3H hybrid background at 3 months of age. Nuclear was stained with Hematoxylin. (A) Coronal forebrain sections from wild-type (WT) and Ts1 mice. Scale bars: 1 mm. (B, C) Magnified images of the boxed areas in A. Scale bars: 50 μm. CC, corpus callosum; STR, striatum. (D) Coronal sections of hippocampal DG from WT and Ts1 mice. Scale bar: 50 μm. (E) Magnified images of the boxed areas in D. Scale bars: 50 μm. GCL, granule cell layer; SGL, subgranular layer (2–3 cells widths along the border of the GCL and hilus). (F) Quantification of DCX-positive cells in the SVZ and DG (GSL and SGL) by counting in a blinded manner shows that the number of DCX-positive neuroblasts is reduced in the SVZ and DG of Ts1 compared with WT littermates. The average number of DCX-positive cells in WT mice was set equal to 100. Each bar corresponds to the total number of DCX-positive cells per a LV wall or a DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test. **P < 0.01 significantly different from WT littermate.

Figure 4.

Decreased DCX-positive neuroblasts in the SVZ and DG of Ts1Cje. Immunohistochemistry using anti- DCX antibody was performed on the brain sections from Ts1Cje (Ts1) mice on the B6/C3H hybrid background at 3 months of age. Nuclear was stained with Hematoxylin. (A) Coronal forebrain sections from wild-type (WT) and Ts1 mice. Scale bars: 1 mm. (B, C) Magnified images of the boxed areas in A. Scale bars: 50 μm. CC, corpus callosum; STR, striatum. (D) Coronal sections of hippocampal DG from WT and Ts1 mice. Scale bar: 50 μm. (E) Magnified images of the boxed areas in D. Scale bars: 50 μm. GCL, granule cell layer; SGL, subgranular layer (2–3 cells widths along the border of the GCL and hilus). (F) Quantification of DCX-positive cells in the SVZ and DG (GSL and SGL) by counting in a blinded manner shows that the number of DCX-positive neuroblasts is reduced in the SVZ and DG of Ts1 compared with WT littermates. The average number of DCX-positive cells in WT mice was set equal to 100. Each bar corresponds to the total number of DCX-positive cells per a LV wall or a DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test. **P < 0.01 significantly different from WT littermate.

Figure 5.

Decreased DCX-positive neuroblasts in the SVZ and DG of Ts2Cje. DCX-positive cells were detected in SVZ (AC) and DG (D, E) of Ts2Cje (Ts2) mice on the B6/C3H hybrid background at 3 months of age. (A) Coronal forebrain sections from Ts2 and their wild-type (WT) littermate. Scale bars: 1 mm. (B, C) Magnified images of the boxed areas in A. Scale bars: 50 μm. (D) Coronal sections of DG from WT and Ts2. Scale bar: 50 μm. (E) Magnified images of the boxed areas in D. Scale bar: 50 μm. (F) Quantification of DCX-positive cells in SVZ and DG by counting in a blinded manner shows that the number of DCX-positive neuroblasts are reduced in Ts2 similarly to that in Ts1Cje. Each value corresponds to relative number of DCX-positive cells against the average number of WT (mean ± standard error of the mean). Statistical significance was determined with the Student's t-test (n = 3 in each genotype). ***P < 0.001 significantly different from WT littermate.

Figure 5.

Decreased DCX-positive neuroblasts in the SVZ and DG of Ts2Cje. DCX-positive cells were detected in SVZ (AC) and DG (D, E) of Ts2Cje (Ts2) mice on the B6/C3H hybrid background at 3 months of age. (A) Coronal forebrain sections from Ts2 and their wild-type (WT) littermate. Scale bars: 1 mm. (B, C) Magnified images of the boxed areas in A. Scale bars: 50 μm. (D) Coronal sections of DG from WT and Ts2. Scale bar: 50 μm. (E) Magnified images of the boxed areas in D. Scale bar: 50 μm. (F) Quantification of DCX-positive cells in SVZ and DG by counting in a blinded manner shows that the number of DCX-positive neuroblasts are reduced in Ts2 similarly to that in Ts1Cje. Each value corresponds to relative number of DCX-positive cells against the average number of WT (mean ± standard error of the mean). Statistical significance was determined with the Student's t-test (n = 3 in each genotype). ***P < 0.001 significantly different from WT littermate.

We also investigated the incorporation of the thymidine analog BrdU (Clark et al. 2006) into the SVZ and DG in Ts1Cje and Ts2Cje on the B6/C3H background at 3 months of age (See Materials and Methods for the protocol). At 24 hours after the last BrdU injection, the BrdU-positive cells in the SVZ and DG of Ts1Cje and Ts2Cje were immunohistochemically stained and counted in a blinded manner (Fig. 6A–F for Ts1Cje and Fig. 7A–F for Ts2Cje). The numbers of BrdU-positive cells in the SVZ and DG of Ts1Cje mice were significantly reduced, by approximately 40% and 35%, respectively, compared with wild-type littermates (Fig. 6G). Similarly, a decreased number of BrdU-positive cells was detected in the SVZ and DG of Ts2Cje (Fig. 7G).

Figure 6.

Decreased BrdU-positive proliferating cells in SVZ and DG of Ts1Cje. Wild-type (WT) and Ts1Cje mice (Ts1) on the B6/C3H hybrid background at 3 months of age were labeled with BrdU (see Materials and Methods). The BrdU-positive cells in SVZ (AD) and DG (E, F) were detected by immunohistochemistry at 24 h after last BrdU injection. Nuclear was stained with Hematoxylin. (A) Coronal forebrain sections from WT and Ts1. Scale bars: 1 mm. (B) Magnified images of the boxed area in A. Scale bars: 1 mm. (C, D) Magnified images of the boxed area in B. The boxed areas surrounded by red and black line in B correspond to C and D, respectively. Scale bars: 50 μm. (E) Coronal sections of DG from WT and Ts1. Scale bar: 100 μm. (F) Magnified images of the boxed area in E. Scale bar: 25 μm. (G) Quantification of BrdU-positive cells by counting in a blinded manner shows that the number of BrdU-labeled proliferating cells is decreased in SVZ and DG (granular cell layer [GSL] and subgranular cell layer [SGL]) of Ts1 compared with that of WT littermates. The averaged number of BrdU-positive cells in the WT littermates was set equal to 100. Each bar corresponds to the total number of BrdU-positive nuclei per LV wall or DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test. *P < 0.05, **P < 0.01 significantly different from WT littermate.

Figure 6.

Decreased BrdU-positive proliferating cells in SVZ and DG of Ts1Cje. Wild-type (WT) and Ts1Cje mice (Ts1) on the B6/C3H hybrid background at 3 months of age were labeled with BrdU (see Materials and Methods). The BrdU-positive cells in SVZ (AD) and DG (E, F) were detected by immunohistochemistry at 24 h after last BrdU injection. Nuclear was stained with Hematoxylin. (A) Coronal forebrain sections from WT and Ts1. Scale bars: 1 mm. (B) Magnified images of the boxed area in A. Scale bars: 1 mm. (C, D) Magnified images of the boxed area in B. The boxed areas surrounded by red and black line in B correspond to C and D, respectively. Scale bars: 50 μm. (E) Coronal sections of DG from WT and Ts1. Scale bar: 100 μm. (F) Magnified images of the boxed area in E. Scale bar: 25 μm. (G) Quantification of BrdU-positive cells by counting in a blinded manner shows that the number of BrdU-labeled proliferating cells is decreased in SVZ and DG (granular cell layer [GSL] and subgranular cell layer [SGL]) of Ts1 compared with that of WT littermates. The averaged number of BrdU-positive cells in the WT littermates was set equal to 100. Each bar corresponds to the total number of BrdU-positive nuclei per LV wall or DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test. *P < 0.05, **P < 0.01 significantly different from WT littermate.

Figure 7.

Decreased BrdU-positive proliferating cells in SVZ and DG of Ts2Cje. BrdU-positive cells were detected in wild-type (WT) and Ts2Cje mice (Ts2) on the B6/C3H hybrid background at 3 months of age. (A) Coronal forebrain sections from Ts2 and their WT littermates. Scale bars: 1 mm. (B) Magnified images of the boxed area in A. Scale bars: 1 mm. (C, D) Magnified images of the boxed area in B. The boxed areas surrounded by red and black line in B correspond to C and D, respectively. Scale bars: 50 μm. (E) Coronal sections of DG from WT and Ts2. Scale bar: 100 μm. (F) Magnified images of the boxed area in E. Scale bar: 25 μm. (G) Quantification of BrdU-positive cells by counting in a blinded manner shows that the number of BrdU-labeled proliferating cells is decreased in SVZ and DG (granular cell layer [GSL] and subgranular cell layer [SGL]) of Ts2 compared with that of WT littermates. The averaged number of BrdU-positive cells in the WT littermates was set equal to 100. Each bar corresponds to the total number of BrdU-positive nuclei per a LV wall or a DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test (n = 3 in each genotype). **P < 0.01 significantly different from WT littermate.

Figure 7.

Decreased BrdU-positive proliferating cells in SVZ and DG of Ts2Cje. BrdU-positive cells were detected in wild-type (WT) and Ts2Cje mice (Ts2) on the B6/C3H hybrid background at 3 months of age. (A) Coronal forebrain sections from Ts2 and their WT littermates. Scale bars: 1 mm. (B) Magnified images of the boxed area in A. Scale bars: 1 mm. (C, D) Magnified images of the boxed area in B. The boxed areas surrounded by red and black line in B correspond to C and D, respectively. Scale bars: 50 μm. (E) Coronal sections of DG from WT and Ts2. Scale bar: 100 μm. (F) Magnified images of the boxed area in E. Scale bar: 25 μm. (G) Quantification of BrdU-positive cells by counting in a blinded manner shows that the number of BrdU-labeled proliferating cells is decreased in SVZ and DG (granular cell layer [GSL] and subgranular cell layer [SGL]) of Ts2 compared with that of WT littermates. The averaged number of BrdU-positive cells in the WT littermates was set equal to 100. Each bar corresponds to the total number of BrdU-positive nuclei per a LV wall or a DG (mean ± standard error of the mean, n = 3 in each genotype). Statistical significance was determined with the Student's t-test (n = 3 in each genotype). **P < 0.01 significantly different from WT littermate.

The olfactory bulb granular and periglomerular layers are supplied with newly generated neurons originating from the SVZ in the mouse brain (Altman 1969). We therefore investigated the volumes of olfactory bulbs of Ts1Cje and Ts2Cje mice on the B6/C3H hybrid background using horizontal MRI images. We found that the olfactory bulbs of both strains of mice were significantly smaller than those of wild-type littermates (Supplementary Fig. S5), possibly reflecting the impaired adult neurogenesis in SVZ of these DS models. Taken together, these results indicate that the postnatal neurogenesis is impaired to a similar degree in both the SVZ and DG of these DS mouse models.

Impaired Embryonic Neurogenesis in Ts1Cje and Ts2Cje Neocortices

We further examined prenatal neurogenesis in the neocortices of Ts1Cje and Ts2Cje. Pregnant Ts1Cje and Ts2Cje females on the B6/C3H hybrid background were injected with BrdU at gestational day 13 (E13.5 for embryos), and the embryos were killed at 24 h later. Although no ventricular enlargement was detected in Ts1Cje and Ts2Cje embryos, the brains of these DS mouse models were significantly smaller than those of their respective wild-type littermates (Fig. 8A, B), suggesting that brain development is disturbed in these DS model mice. Concomitantly, the cortical thicknesses in these DS mice were less than those of their wild-type littermates (Fig. 8C). In addition, the ratios of the total number of BrdU-positive cells throughout the entire thickness of dorsal pallium were significantly lower in the DS model mice compared with those of respective wild-type littermates (Fig. 8C, D). In each cortical area, VZ/SVZ, IZ, and SP/CP, a similar reduction in BrdU-positive cells was observed in Ts1Cje and Ts2Cje mice (Fig. 8D). We further confirmed the reduction of BrdU-positive cells in these DS models by measurement of immunopositive area with NIH ImageJ software and similarly reduced ratios of the immunopositive area/total cortical area were observed in both DS models compared with those of respective wild-type littermates (Fig. 8E). Next, we performed double-immunofluorescence labellings on these brain sections with antibodies against BrdU and Ki67, a protein marker for proliferating cells. Ki67-negative cells in the BrdU-positive cell cohort correspond to cells that have exited the cell cycle and differentiated in the period between E13.5 and E14.5 (Fig. 9A, B). The ratios of BrdU-positive cells/all cells or BrdU-positive, Ki67-negative cells/all cells in Ts1Cje and Ts2Cje, counted by the stereological method (Williams and Rakic 1988) on fluorescence microscope with z-axis controller in a blinded manner, were lower than that of respective wild-type littermates (Fig. 9C, D). These results suggest that prenatal neurogenesis is also impaired in both Ts1Cje and Ts2Cje mice at a similar degree.

Figure 8.

Decreased proliferating cells in Ts1Cje and Ts2Cje embryonic neocortices. Pregnant Ts1Cje (Ts1) and Ts2Cje (Ts2) females on the B6/C3H hybrid background at gestational day 13 (E13.5 for embryos) were administrated with one pulse of 50 mg/kg BrdU and BrdU-positive cells were detected at 24 h after injection on immunohistochemistry. (A) BrdU immunostaining of coronal sections from Ts1 (left) and Ts2 (right) taken from matched sections according to the morphology of somatosensory cortex. Scale bar: 1 mm. (B) Areas of entire brains from Ts1, Ts2, and respective wild-type (WT) littermates (n = 4 for pairs of Ts1 and n = 3 for pairs of Ts2) were measured by NIH ImageJ software. Each value indicates area of the entire brain in arbitrary unit (mean ± standard error of the mean). Note that the brains of Ts1 and Ts2 were significantly smaller than those of respective WT littermates. (C) Higher magnification images of the cortical wall taken at the midpoint between the medial and lateral angles of the LV (boxed areas in A) show that pallial thickness of DS models is thinner than that of respective WT littermate. In addition, fewer BrdU-positive cells (brown) were detected in both DS models. Nuclear was stained with Hematoxylin (blue). Scale bar: 50 μm. (D) Higher magnification images of each layer. Scale bar: 25 μm. (E, F) Quantifications of BrdU-positive and -negative cell numbers (D) and their areas (E) in each layer (VZ/SVZ, IZ, and SP/CP) of the cortex by counting in a blinded manner using NIH ImageJ. Proliferating cells were reduced in embryonic cortices of both Ts1 and Ts2. Values in D and E indicate the ratios of the number of BrdU-positive cells/total cells and of the BrdU-positive area/total area, respectively (mean ± standard error of the mean). Statistical significance was determined with the Student's t-test. *P < 0.05, significantly different from WT littermate.

Figure 8.

Decreased proliferating cells in Ts1Cje and Ts2Cje embryonic neocortices. Pregnant Ts1Cje (Ts1) and Ts2Cje (Ts2) females on the B6/C3H hybrid background at gestational day 13 (E13.5 for embryos) were administrated with one pulse of 50 mg/kg BrdU and BrdU-positive cells were detected at 24 h after injection on immunohistochemistry. (A) BrdU immunostaining of coronal sections from Ts1 (left) and Ts2 (right) taken from matched sections according to the morphology of somatosensory cortex. Scale bar: 1 mm. (B) Areas of entire brains from Ts1, Ts2, and respective wild-type (WT) littermates (n = 4 for pairs of Ts1 and n = 3 for pairs of Ts2) were measured by NIH ImageJ software. Each value indicates area of the entire brain in arbitrary unit (mean ± standard error of the mean). Note that the brains of Ts1 and Ts2 were significantly smaller than those of respective WT littermates. (C) Higher magnification images of the cortical wall taken at the midpoint between the medial and lateral angles of the LV (boxed areas in A) show that pallial thickness of DS models is thinner than that of respective WT littermate. In addition, fewer BrdU-positive cells (brown) were detected in both DS models. Nuclear was stained with Hematoxylin (blue). Scale bar: 50 μm. (D) Higher magnification images of each layer. Scale bar: 25 μm. (E, F) Quantifications of BrdU-positive and -negative cell numbers (D) and their areas (E) in each layer (VZ/SVZ, IZ, and SP/CP) of the cortex by counting in a blinded manner using NIH ImageJ. Proliferating cells were reduced in embryonic cortices of both Ts1 and Ts2. Values in D and E indicate the ratios of the number of BrdU-positive cells/total cells and of the BrdU-positive area/total area, respectively (mean ± standard error of the mean). Statistical significance was determined with the Student's t-test. *P < 0.05, significantly different from WT littermate.

Figure 9.

Decreased BrdU-positive cells exiting the cell cycle in Ts1Cje and Ts2Cje embryonic neocortices. Pregnant Ts1Cje (Ts1) and Ts2Cje (Ts2) females on the B6/C3H hybrid background at gestational day 13 (E13.5 for embryos) were administrated with one pulse of 50 mg/kg BrdU, and BrdU- and Ki67-positive cells were detected at 24 h after injection on immunofluorescence. (A) Double-staining images of the cortical wall for Ki67 (green) and BrdU (red) taken at the midpoint between the medial and lateral angles of the LV show that fewer BrdU-positive and Ki67-negative, BrdU-positive cells (exiting the cell cycle) were detected in both DS models. Nuclear was stained with DAPI. Scale bar: 75 μm. (B) Higher magnification images of each layer. Scale bar: 50 μm. (C, D) Percentages of the number of BrdU-positive (C) and Ki67-negative/BrdU-positive cells (D) against total cell numbers (on the DAPI staining) in each layer (VZ/SVZ, IZ, and SP/CP) of the cortex were calculated by cell counting with stereological method. Number of cells with proliferating (C) and exiting cell cycle (D) were reduced in embryonic cortices of both Ts1 and Ts2. Values in C and D indicate the ratios of BrdU(+) cell number/total cell number and BrdU(+), Ki67(−) cell number/total cell number, respectively (n = 2 pairs, mean ± standard error of the mean). Statistical significance was determined with the Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 significantly different from WT littermate.

Figure 9.

Decreased BrdU-positive cells exiting the cell cycle in Ts1Cje and Ts2Cje embryonic neocortices. Pregnant Ts1Cje (Ts1) and Ts2Cje (Ts2) females on the B6/C3H hybrid background at gestational day 13 (E13.5 for embryos) were administrated with one pulse of 50 mg/kg BrdU, and BrdU- and Ki67-positive cells were detected at 24 h after injection on immunofluorescence. (A) Double-staining images of the cortical wall for Ki67 (green) and BrdU (red) taken at the midpoint between the medial and lateral angles of the LV show that fewer BrdU-positive and Ki67-negative, BrdU-positive cells (exiting the cell cycle) were detected in both DS models. Nuclear was stained with DAPI. Scale bar: 75 μm. (B) Higher magnification images of each layer. Scale bar: 50 μm. (C, D) Percentages of the number of BrdU-positive (C) and Ki67-negative/BrdU-positive cells (D) against total cell numbers (on the DAPI staining) in each layer (VZ/SVZ, IZ, and SP/CP) of the cortex were calculated by cell counting with stereological method. Number of cells with proliferating (C) and exiting cell cycle (D) were reduced in embryonic cortices of both Ts1 and Ts2. Values in C and D indicate the ratios of BrdU(+) cell number/total cell number and BrdU(+), Ki67(−) cell number/total cell number, respectively (n = 2 pairs, mean ± standard error of the mean). Statistical significance was determined with the Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 significantly different from WT littermate.

Discussion

In this study, we showed enlargement of the ventricles of the brain and impaired neurogenesis in 2 DS mouse models, Ts1Cje and Ts2Cje (Table 1). Although ventricular enlargement has been described in DS (Pearlson et al. 1998; White et al. 2003; Schimmel et al. 2006), to our knowledge this is the first report in DS mouse models. Ventricular enlargement in Ts65Dn has not been reported in any studies so far (Table 1); however, we assume that this would be also the case in Ts65Dn that is genetically equivalent to Ts2Cje. Impairment of prenatal and postnatal neurogenesis has been shown in the hippocampal DG and neocortex of Ts65Dn mouse, respectively (Clark et al. 2006; Chakrabarti et al. 2007) (Table 1). We have confirmed it in the equivalent mouse model, Ts2Cje, and have also demonstrated similar impairment in the postnatal SVZ. Furthermore, for the first time, we have found similar defects in postnatal and prenatal neurogenesis in another DS model, Ts1Cje, in which the trisomic segment corresponds to two-thirds of the trisomic segment in Ts2Cje.

The enlargement of brain ventricles has been described for various brain disorders with cognitive impairment, including multiple sclerosis (Bakshi et al. 2002), first-episode schizophrenia (Fannon et al. 2000), Klinefelter syndrome (a sex chromosome aneuploidy) (Itti et al. 2006), non-DS mental retardation (Spencer et al. 2005), fragile X mental retardation (Reiss et al. 1995), and periventricular leukomalacia (Melhem et al. 2000). Furthermore, several studies have described correlations between the degrees of ventricular enlargement and the level of the cognitive dysfunction or mental retardation (Reiss et al. 1995; Melhem et al. 2000; Spencer et al. 2005). The enlargements of the ventricles in the 2 DS models we have studied are very similar to one another, but the learning and memory defects are more severe in Ts65Dn and the equivalent Ts2Cje, in comparison to Ts1Cje (Sago et al. 2000). This difference of cognitive severity may be the result of the extra trisomic segment that is specific to Ts2Cje/Ts65Dn. For example, Ts65Dn mice, but not Ts1Cje, exhibit age-dependent atrophy of basal forebrain cholinergic neurons (BFCNs) (Holtzman et al. 1996; Sago et al. 1998) and increased expression of the amyloid-beta precursor protein gene (App), which is located on the Ts65Dn trisomic segment but not on that of Ts1Cje, has been reported to be responsible for the degeneration of BFCNs (Salehi et al. 2006). App could be one of such factors that are responsible for the Ts65Dn-specific portion of learning defects.

What is the underlying mechanism for the ventricular enlargement in DS mouse models? Although obstruction of fourth ventricle and/or cerebral aqueduct has been known to cause ventricular expansion (Garton and Piatt 2004), this was not the case in both Ts1Cje and Ts2Cje. Enlargement of the ventricular system may also be caused by a failure of absorption or an overproduction of CSF, structural or functional impairments of cilia, and impaired cell proliferation around ventricles (Garton and Piatt 2004). Among these possibilities, impaired cell proliferation in the DS model brains would be the most plausible. Retardation of prenatal brain development in Ts16 and Ts65Dn mice and reduced embryonic neurogenesis in Ts65Dn have been demonstrated (Haydar et al. 1996, 2000; Chakrabarti et al. 2007). These and our present observations allow us to hypothesize that impaired prenatal neurogenesis may result in an impairment of prenatal brain development and the expansion of brain ventricles postnatally in model mice with DS.

It has been proposed that some symptoms of human DS, such as developmental delay, deficits in intellectual function, and occasional seizures, are based on abnormalities in cortical dysgenesis associated with prenatal defects of neurogenesis or neuronal migration and impaired pre- and postnatal synaptogenesis (Wisniewski et al. 1984). Impaired cortical neurogenesis and development at prenatal stage would be expected to result in abnormal defects in the postnatal connections between the cortex and other brain regions. Such defects in connectivity could lead to cognitive dysfunction and mental retardation in individuals with DS.

It has also been shown that adult-generated neurons in DG have the potential to become synaptically integrated (Markakis and Gage 1999; Carlén et al. 2002) and to attain neuronal characteristics morphologically, biochemically, and electrophysiologically (van Praag et al. 2002; Schmidt-Hieber et al. 2004; Kee et al. 2007). The genetic ablation of newly formed neurons in adult mice has resulted in defects in the retention of spatial memory (Imayoshi et al. 2008). Thus, adult neurogenesis in the DG is suggested to play a role on spatial memory and learning in rodents, and its decline may also contribute to the cognitive defects.

Overexpressions of the dual specificity tyrosine-regulated kinase 1a gene DYRK1A on human chromosome 21 or Dyrk1a on the Ts1Cje trisomic segment have been proposed to be responsible for the decreased transcript levels of neuron-restrictive silencer factor (NRSF/REST) and its target molecules such as Nanog and Sox2 in DS mouse models and patients (Canzonetta et al. 2008). Interestingly, Sox2 deficiency has been reported to cause ventricular enlargements and impairment of neurogenesis in the adult mouse brain (Ferri et al. 2004). Dyrk1a would therefore be one of plausible candidate genes responsible for the ventricle enlargements and impaired neurogenesis observed in Ts1Cje and Ts2Cje and would link these 2 abnormal parameters. Another gene on the Ts1Cje trisomic segment, Olig2, would also be an interesting candidate. The Olig2 null mice lacks oligodendrocytes and motor neurons in the spinal cord (Lu et al. 2002). Importantly, Olig2 opposes the neurogenic role of the box 6 protein (PAX6) and promotes oligodendrogenesis (Hack et al. 2005). PAX6 is a factor essential for production and maintenance of the early progenitor cells in the postnatal hippocampal neurogenesis (Maekawa et al. 2005). The overexpression of Olig2 could therefore disturb the neurogenesis in DS mouse models and patients.

In summary, we have shown ventricular enlargement and impaired neurogenesis in the brains of 2 DS mouse models, Ts1Cje and Ts2Cje. These abnormal phenotypes were of a similar degree in the 2 models, even though the trisomic segment of Ts2Cje mice is longer than that of Ts1Cje mice. These results suggest that the genes responsible for the enlargement of brain ventricles and impaired neurogenesis is located on the Ts1Cje trisomic segment. The identification and characterization of these genes should contribute to elucidate the relationship between these abnormalities and memory and learning deficits in DS mouse models and may lead to a further understanding of the molecular pathology of DS mental retardation.

Table 1

Comparison among DS mouse models on ventricular enlargement and impaired neurogenesis

 Ts1Cje Ts2Cje Ts65Dn 
Enlargement of LV s on MRI 200–400% 200–300% n.a.a 
DCX-positive cells in 3-month-old adult mice SVZ DG SVZ DG SVZ DG 
70% 40% 60% 50% n.a.a n.a.a 
BrdU-positive cells in 3-month-old adult mice SVZ DG SVZ DG SVZ DG 
60% 60% 60% 60% n.a.a 50% (Clark et al., 2006
BrdU-positive cells in embryonic stage (cortical proliferating cells) 75%b 70%b 50%b (Chakrabarti et al. 2007) 
 Ts1Cje Ts2Cje Ts65Dn 
Enlargement of LV s on MRI 200–400% 200–300% n.a.a 
DCX-positive cells in 3-month-old adult mice SVZ DG SVZ DG SVZ DG 
70% 40% 60% 50% n.a.a n.a.a 
BrdU-positive cells in 3-month-old adult mice SVZ DG SVZ DG SVZ DG 
60% 60% 60% 60% n.a.a 50% (Clark et al., 2006
BrdU-positive cells in embryonic stage (cortical proliferating cells) 75%b 70%b 50%b (Chakrabarti et al. 2007) 
a

n.a., data not available.

b

We counted all BrdU-positive cells on immunohistochemistry. In contrast, Chakrabarti et al. counted only heavy labeled cells.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

Grant from RIKEN Brain Science Institute (to K.Y.); Grant-in-aid for Scientific Research (Project 20790089 to K.I.), the Ministry of Education, Science, Culture and Sports of Japan.

We are grateful to Drs Takeuchi T. and Suzuki T., Laboratory for Neurogenetics, RIKEN, BSI, Japan, for the technical assistance. Conflict of Interest: None declared.

References

Altman
J
Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb
J Comp Neurol
 , 
1969
, vol. 
137
 (pg. 
433
-
457
)
Amano
K
Sago
H
Uchikawa
C
Suzuki
T
Kotliarova
SE
Nukina
N
Epstein
CJ
Yamakawa
K
Dosage-dependent over-expression of genes in the trisomic region of Ts1Cje mouse model for Down syndrome
Hum Mol Genet.
 , 
2004
, vol. 
13
 (pg. 
1333
-
1340
)
Aylward
EH
Li
Q
Honeycutt
NA
Warren
AC
Pulsifer
MB
Barta
PE
Chan
MD
Smith
PD
Jerram
M
Pearlson
GD
MRI volumes of the hippocampus and amygdala in adults with Down's syndrome with and without dementia
Am J Psychiatry
 , 
1999
, vol. 
156
 (pg. 
564
-
568
)
Bahn
S
Mimmack
M
Ryan
M
Caldwell
MA
Jauniaux
E
Starkey
M
Svendsen
CN
Emson
P
Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study
Lancet
 , 
2002
, vol. 
359
 (pg. 
310
-
315
)
Bakshi
R
Benedict
RH
Bermel
RA
Caruthers
SD
Puli
SR
Tjoa
CW
Fabiano
AJ
Jacobs
L
T2 hypointensity in the deep gray matter of patients with multiple sclerosis: a quantitative magnetic resonance imaging study
Arch Neurol
 , 
2002
, vol. 
59
 (pg. 
62
-
68
)
Belichenko
PV
Kleschevnikov
AM
Salehi
A
Epstein
CJ
Mobley
WC
Synaptic and cognitive abnormalities in mouse models of Down syndrome: exploring genotype-phenotype relationships
J Comp Neurol
 , 
2007
, vol. 
504
 (pg. 
329
-
345
)
Belichenko
PV
Masliah
E
Kleschevnikov
AM
Villar
AJ
Epstein
CJ
Salehi
A
Mobley
WC
Synaptic structural abnormalities in the Ts65Dn mouse model of Down syndrome
J Comp Neurol
 , 
2004
, vol. 
480
 (pg. 
281
-
298
)
Brown
JP
Couillard-Despres
S
Cooper-Kuhn
CM
Winkler
J
Aigner
L
Kuhn
HG
Transient expression of doublecortin during adult neurogenesis
J Comp Neurol
 , 
2003
, vol. 
467
 (pg. 
1
-
10
)
Canzonetta
C
Mulligan
C
Deutsch
S
Ruf
S
O'Doherty
A
Lyle
R
Borel
C
Lin-Marq
N
Delom
F
Groet
J
, et al.  . 
DYRK1A-dosage imbalance perturbs NRSF/REST levels, deregulating pluripotency and embryonic stem cell fate in Down syndrome
Am J Hum Genet
 , 
2008
, vol. 
83
 (pg. 
388
-
400
)
Carlén
M
Cassidy
RM
Brismar
H
Smith
GA
Enquist
LW
Frisen
J
Functional integration of adult-born neurons
Curr Biol
 , 
2002
, vol. 
12
 (pg. 
606
-
608
)
Chakrabarti
L
Galdzicki
Z
Haydar
TF
Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome
J Neurosci
 , 
2007
, vol. 
27
 (pg. 
11483
-
11495
)
Clark
S
Schwalbe
J
Stasko
MR
Yarowsky
PJ
Costa
AC
Fluoxetine rescues deficient neurogenesis in hippocampus of the Ts65Dn mouse model for Down syndrome
Exp Neurol
 , 
2006
, vol. 
200
 (pg. 
256
-
261
)
Contestabile
A
Fila
T
Ceccarelli
C
Bonasoni
P
Bonapace
L
Santini
D
Bartesaghi
R
Ciani
E
Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice
Hippocampus
 , 
2007
, vol. 
17
 (pg. 
665
-
678
)
Dauphinot
L
Lyle
R
Rivals
I
Dang
MT
Moldrich
RX
Golfier
G
Ettwiller
L
Toyama
K
Rossier
J
Personnaz
L
, et al.  . 
The cerebellar transcriptome during postnatal development of the Ts1Cje mouse, a segmental trisomy model for Down syndrome
Hum Mol Genet
 , 
2005
, vol. 
14
 (pg. 
373
-
384
)
Davisson
MT
Schmidt
C
Akeson
EC
Segmental trisomy of murine chromosome 16: a new model system for studying Down syndrome
Prog Clin Biol Res.
 , 
1990
, vol. 
360
 (pg. 
263
-
280
)
Epstein
CJ
Scriver
CR
Beaudet
AL
Sly
WS
Valle
D
Down syndrome (trisomy 21)
The metabolic and molecular bases inherited disease
 , 
2001
8th ed
New York
McGraw Hill
(pg. 
1223
-
1256
)
Fannon
D
Tennakoon
L
Sumich
A
O'Ceallaigh
S
Doku
V
Chitnis
X
Lowe
J
Soni
W
Sharma
T
Third ventricle enlargement and developmental delay in first-episode psychosis: preliminary findings
Br J Psychiatry
 , 
2000
, vol. 
177
 (pg. 
354
-
359
)
Ferri
AL
Cavallaro
M
Braida
D
Di Cristofano
A
Canta
A
Vezzani
A
Ottolenghi
S
Pandolfi
PP
Sala
M
DeBiasi
S
, et al.  . 
Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain
Development
 , 
2004
, vol. 
131
 (pg. 
3805
-
3819
)
Garton
HJ
Piatt
JH
Jr
Hydrocephalus
Pediatr Clin North Am
 , 
2004
, vol. 
51
 (pg. 
305
-
325
)
Golden
JA
Hyman
BT
Development of the superior temporal neocortex is anomalous in trisomy 21
J Neuropathol Exp Neurol
 , 
1994
, vol. 
53
 (pg. 
513
-
520
)
Hack
MA
Saghatelyan
A
de Chevigny
A
Pfeifer
A
Ashery-Padan
R
Lledo
PM
Götz
M
Neuronal fate determinants of adult olfactory bulb neurogenesis
Nat Neurosci
 , 
2005
, vol. 
8
 (pg. 
865
-
872
)
Haydar
TF
Blue
ME
Molliver
ME
Krueger
BK
Yarowsky
PJ
Consequences of trisomy 16 for mouse brain development: corticogenesis in a model of Down syndrome
J Neurosci
 , 
1996
, vol. 
16
 (pg. 
6175
-
6182
)
Haydar
TF
Nowakowski
RS
Yarowsky
PJ
Krueger
BK
Role of founder cell deficit and delayed neuronogenesis in microencephaly of the trisomy 16 mouse
J Neurosci
 , 
2000
, vol. 
20
 (pg. 
4156
-
4164
)
Holtzman
DM
Santucci
D
Kilbridge
J
Chua-Couzens
J
Fontana
DJ
Daniels
SE
Johnson
RM
Chen
K
Sun
Y
Carlson
E
, et al.  . 
Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome
Proc Natl Acad Sci USA
 , 
1996
, vol. 
93
 (pg. 
13333
-
13338
)
Imayoshi
I
Sakamoto
M
Ohtsuka
T
Takao
K
Miyakawa
T
Yamaguchi
M
Mori
K
Ikeda
T
Itohara
S
Kageyama
R
Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain
Nat Neurosci
 , 
2008
, vol. 
11
 (pg. 
1153
-
1161
)
Itti
E
Gaw Gonzalo
IT
Pawlikowska-Haddal
A
Boone
KB
Mlikotic
A
Itti
L
Mishkin
FS
Swerdloff
RS
The structural brain correlates of cognitive deficits in adults with Klinefelter's syndrome
J Clin Endocrinol Metab
 , 
2006
, vol. 
91
 (pg. 
1423
-
1427
)
JAX Notes
Down syndrome model distribution expanded [Internet]. JAX Notes
 , 
2005
 
Bar Harbor (ME): Jackson Laboratory. Available from: http://jaxmice.jax.org/jaxnotes/archive/497b.html
Kahlem
P
Sultan
M
Herwig
R
Steinfath
M
Balzereit
D
Eppens
B
Saran
NG
Pletcher
MT
South
ST
Stetten
G
, et al.  . 
Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of Down syndrome
Genome Res
 , 
2004
, vol. 
14
 (pg. 
1258
-
1267
)
Kee
N
Teixeira
CM
Wang
AH
Frankland
PW
Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus
Nat Neurosci
 , 
2007
, vol. 
10
 (pg. 
355
-
362
)
Kesslak
JP
Nagata
SF
Lott
I
Nalcioglu
O
Magnetic resonance imaging analysis of age-related changes in the brains of individuals with Down's syndrome
Neurology
 , 
1994
, vol. 
44
 (pg. 
1039
-
1045
)
Kleschevnikov
AM
Belichenko
PV
Villar
AJ
Epstein
CJ
Malenka
RC
Mobley
WC
Hippocampal long-term potentiation suppressed by increased inhibition in the Ts65Dn mouse, a genetic model of Down syndrome
J Neurosci
 , 
2004
, vol. 
15
 (pg. 
8153
-
8160
)
Lockrow
J
Prakasam
A
Huang
P
Bimonte-Nelson
H
Sambamurti
K
Granholm
AC
Cholinergic degeneration and memory loss delayed by vitamin E in a Down syndrome mouse model
Exp Neurol
 , 
2009
, vol. 
216
 (pg. 
278
-
289
)
Lu
QR
Sun
T
Zhu
Z
Ma
N
Garcia
M
Stiles
CD
Rowitch
DH
Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection
Cell
 , 
2002
, vol. 
109
 (pg. 
75
-
86
)
Maekawa
M
Takashima
N
Arai
Y
Nomura
T
Inokuchi
K
Yuasa
S
Osumi
N
Pax6 is required for production and maintenance of progenitor cells in postnatal hippocampal neurogenesis
Genes Cells
 , 
2005
, vol. 
10
 (pg. 
1001
-
1014
)
Markakis
EA
Gage
FH
Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles
J Comp Neurol
 , 
1999
, vol. 
406
 (pg. 
449
-
460
)
Melhem
ER
Hoon
AH
Jr
Ferrucci
JT
Jr
Quinn
CB
Reinhardt
EM
Demetrides
SW
Freeman
BM
Johnston
MV
Periventricular leukomalacia: relationship between lateral ventricular volume on brain MR images and severity of cognitive and motor impairment
Radiology
 , 
2000
, vol. 
214
 (pg. 
199
-
204
)
Paxinos
G
Franklin
K
The mouse brain in stereotaxic coordinates
 , 
2001
2nd ed
San Diego
Academic Press
Pearlson
GD
Breiter
SN
Aylward
EH
Warren
AC
Grygorcewicz
M
Frangou
S
Barta
PE
Pulsifer
MB
MRI brain changes in subjects with Down syndrome with and without dementia
Dev Med Child Neurol
 , 
1998
, vol. 
40
 (pg. 
326
-
334
)
Raz
N
Torres
IJ
Briggs
SD
Spencer
WD
Thornton
AE
Loken
WJ
Gunning
FM
McQuain
JD
Driesen
NR
Acker
JD
Selective neuroanatomic abnormalities in Down's syndrome and their cognitive correlates: evidence from MRI morphometry
Neurology
 , 
1995
, vol. 
45
 (pg. 
356
-
366
)
Reeves
RH
Irving
NG
Moran
TH
Wohn
A
Kitt
C
Sisodia
SS
Schmidt
C
Bronson
RT
Davisson
MT
A mouse model for Down syndrome exhibits learning and behaviour deficits
Nat Genet.
 , 
1995
, vol. 
11
 (pg. 
177
-
184
)
Reiss
AL
Abrams
MT
Greenlaw
R
Freund
L
Denckla
MB
Neurodevelopmental effects of the FMR-1 full mutation in humans
Nat Med
 , 
1995
, vol. 
1
 (pg. 
159
-
167
)
Richtsmeier
JT
Baxter
LL
Reeves
RH
Parallels of craniofacial maldevelopment in Down syndrome and Ts65Dn mice
Dev Dyn
 , 
2000
, vol. 
217
 (pg. 
137
-
145
)
Richtsmeier
JT
Zumwalt
A
Carlson
EJ
Epstein
CJ
Reeves
RH
Craniofacial phenotypes in segmentally trisomic mouse models for Down syndrome
Am J Med Genet
 , 
2002
, vol. 
107
 (pg. 
317
-
324
)
Sago
H
Carlson
EJ
Smith
DJ
Kilbridge
J
Rubin
EM
Mobley
WC
Epstein
CJ
Huang
TT
Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities
Proc Natl Acad Sci USA
 , 
1998
, vol. 
95
 (pg. 
6256
-
6261
)
Sago
H
Carlson
EJ
Smith
DJ
Rubin
EM
Crnic
LS
Huang
TT
Epstein
CJ
Genetic dissection of region associated with behavioral abnormalities in mouse models for Down syndrome
Pediatr Res
 , 
2000
, vol. 
48
 (pg. 
606
-
613
)
Salehi
A
Delcroix
JD
Belichenko
PV
Zhan
K
Wu
C
Valletta
JS
Takimoto-Kimura
R
Kleschevnikov
AM
Sambamurti
K
Chung
PP
, et al.  . 
Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration
Neuron
 , 
2006
, vol. 
51
 (pg. 
29
-
42
)
Schimmel
MS
Hammerman
C
Bromiker
R
Berger
I
Third ventricle enlargement among newborn infants with trisomy 21
Pediatrics
 , 
2006
, vol. 
117
 (pg. 
e928
-
e931
)
Schmidt-Hieber
C
Jonas
P
Bischofberger
J
Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus
Nature
 , 
2004
, vol. 
429
 (pg. 
184
-
187
)
Shukkur
EA
Shimohata
A
Akagi
T
Yu
W
Yamaguchi
M
Murayama
M
Chui
D
Takeuchi
T
Amano
K
Subramhanya
KH
, et al.  . 
Mitochondrial dysfunction and tau hyperphosphorylation in Ts1Cje, a mouse model for Down syndrome
Hum Mol Genet
 , 
2006
, vol. 
15
 (pg. 
2752
-
2762
)
Spencer
MD
Gibson
RJ
Moorhead
TW
Keston
PM
Hoare
P
Best
JJ
Lawrie
SM
Johnstone
EC
Qualitative assessment of brain anomalies in adolescents with mental retardation
AJNR Am J Neuroradiol
 , 
2005
, vol. 
26
 (pg. 
2691
-
2697
)
Takashima
S
Becker
LE
Armstrong
DL
Chan
F
Abnormal neuronal development in the visual cortex of the human fetus and infant with Down's syndrome. A quantitative and qualitative Golgi study
Brain Res.
 , 
1981
, vol. 
225
 (pg. 
1
-
21
)
van Praag
H
Schinder
AF
Christie
BR
Toni
N
Palmer
TD
Gage
FH
Functional neurogenesis in the adult hippocampus
Nature
 , 
2002
, vol. 
415
 (pg. 
1030
-
1034
)
Villar
AJ
Belichenko
PV
Gillespie
AM
Kozy
HM
Mobley
WC
Epstein
CJ
Identification and characterization of a new Down syndrome model, Ts[Rb(12.1716)]2Cje, resulting from a spontaneous Robertsonian fusion between T(1716)65Dn and mouse chromosome 12
Mamm Genome
 , 
2005
, vol. 
16
 (pg. 
79
-
90
)
Weis
S
Weber
G
Neuhold
A
Rett
A
Down syndrome: MR quantification of brain structures and comparison with normal control subjects
AJNR Am J Neuroradiol
 , 
1991
, vol. 
12
 (pg. 
1207
-
1211
)
White
NS
Alkire
MT
Haier
RJ
A voxel-based morphometric study of nondemented adults with Down syndrome
Neuroimage
 , 
2003
, vol. 
20
 (pg. 
393
-
403
)
Williams
RW
Rakic
P
Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material
J Comp Neurol
 , 
1988
, vol. 
278
 (pg. 
344
-
352
)
Wisniewski
KE
Laure-Kamionowska
M
Wisniewski
HM
Evidence of arrest of neurogenesis and synaptogenesis in brains of patients with Down's syndrome
N Engl J Med
 , 
1984
, vol. 
311
 (pg. 
1187
-
1188
)

Author notes

4
Current address: Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan