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As the genomic basis for Down syndrome (DS), human trisomy 21 is the most common genetic cause of intellectual disability in children and young people. The genomic regions on human chromosome 21 (Hsa21) are syntenic to three regions in the mouse genome, located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. Recently, we have developed three new mouse models using chromosome engineering carrying the genotypes of Dp(10)1Yey/+, Dp(16)1Yey/+, or Dp(17)1Yey/+, which harbor a duplication spanning the entire Hsa21 syntenic region on Mmu10, Mmu16, or Mmu17, respectively. In this study, we analyzed the hippocampal long-term potentiation (LTP) and cognitive behaviors of these models. Our results show that, while the genotype of Dp(17)1Yey/+ results in abnormal hippocampal LTP, the genotype of Dp(16)1Yey/+ leads to both abnormal hippocampal LTP and impaired learning/memory. Therefore, these mutant mice can serve as powerful tools for further understanding the mechanism underlying cognitively relevant phenotypes associated with DS, particularly the impacts of different syntenic regions on these phenotypes.
DS, caused by human trisomy 21, is the most frequent live-born human chromosomal disorder (CDC, 2006; Hook et al., 1983; Hook, 1982) and the most common genetic cause of intellectual disability in children and young people (Chapman and Hesketh, 2000; Haxby, 1989; Nadel, 1999; Pennington et al., 2003; Pulsifer, 1996). Evidence has shown that hippocampal dysfunctions are associated with intellectual disability in DS (Pennington et al., 2003; Uecker et al., 1993). The underlying mechanism of this clinical manifestation is not well understood, which hinders the progress of developing any effective therapeutic interventions.
Characterizations of genetically engineered mouse models hold promise for unraveling the molecular mechanisms of DS-associated diseases. The regions on Hsa21 are syntenically conserved with three regions in the mouse genome located on Mmu10, Mmu16 and Mmu17, which contain approximately 41, 115 and 19 Hsa21 gene orthologs, respectively (Fig. 1) (http://www.ensembl.org; http://genome.ucsc.edu). Trans-species mouse models carry Hsa21 or a fragment of it (O'Doherty et al., 2005; Shinohara et al., 2001). Although mosaicism associated with these trans-species models has not been resolved, Tc1 mice, carrying approximately 92% of Hsa21, exhibit impairments in cognitive behaviors and hippocampal LTP, which is considered a major physiological mechanism of learning and memory (Bliss and Collingridge, 1993; Lynch, 2004; Martin et al., 2000; O'Keefe, 1993). Trisomic models carry three copies of segments of mouse syntenic regions of Hsa21. Ts65Dn, the most widely used model (Reeves et al., 1995), is trisomic for 13.4 Mb of the 22.9 Mb Hsa21 syntenic region on Mmu16, which harbors approximately 99 orthologs of Hsa21 genes (Akeson et al., 2001; Kahlem et al., 2004). However, Ts65Dn is also trisomic for a larger-than-5.8 Mb subcentromeric region on Mmu17 that is not syntenic to any region on Hsa21.
To better understand the roles of the different mouse syntenic regions in DS-associated intellectual disability. we have recently developed new mouse models carrying individual duplications spanning the entire Hsa21 syntenic regions on Mmu10, Mmu16, or Mmu17 using chromosome engineering (Yu et al., 2010). In this study, we performed an analysis of the cognitive behaviors and hippocampal LTP of Dp(10)1Yey/+, Dp(16)1Yey/+ and Dp(17)1Yey/+ mice for the first time to assess the impact of the presence of individual duplications on cognitively relevant phenotypes associated with DS.
To examine the effect of individual syntenic regions on hippocampal-mediated learning and memory, we compared the performances between the mutant mice and their wild-type littermates in the Morris water maze tasks. We first examined the mice in the visible platform tests, and there was no statistically significant difference in the latencies or pathlengths in locating the visible platform between the mutant mice and their wild-type littermates. In the hidden platform tests, Dp(10)1Yey/+ mice performed at a similar proficiency to their wild-type littermates (Figs. 2A-2D). On the other hand, we observed Dp(16)1Yey/+ mice took a longer path-length to locate the platform during training trials (p<0.01) (Fig. 2E), although the path-length in locating the platform significantly decreased with training for both the mutant mice and the wild-type littermates (p<0.01). Dp(16)1Yey/+ mice also had a significantly longer average latency in locating the platform (p<0.01) (Fig. 2F). However, the difference in latency may be partially explained by the slower swimming speed of Dp(16)1Yey/+ mice (p<0.01) (Fig. 2G). More importantly, in the probe test performed on the day after the training trials, Dp(16)1Yey/+ mice spent a significantly shorter time in the target quadrant (Northeast, NE) than the wild-type littermates (p<0.05) (Fig. 2H). These data suggest that the genotype of Dp(16)1Yey/+ causes impairment in spatial learning and memory. The performances of Dp(17)1Yey/+ mice are similar to those of their wild-type littermates in the Morris water maze tasks (Figs. 2I-2L), except for the probe test after the learning trials, in which Dp(17)1Yey/+ mice showed a tendency for impairment, although the difference is not statistically significant (p=0.07) (Fig. 2L).
To determine if factors other than spatial learning have contributed to the water maze results, we analyzed the swim-paths of the mutant mice and their wild-type littermates in each quadrant on the first day of the hidden platform Morris water maze test. We found no difference in the path lengths of the mice with any genotype in different quadrants. In addition, we found no difference in the times that the mice spent in different quadrants. These data were collected before any mice had started to learn the location of the platform, indicating there was no bias towards any specific quadrant, including the target quadrant. Therefore, the differences that emerged later in the hidden platform tests are unlikely to be due to factors other than spatial learning.
We also performed a contextual fear conditioning test to assess hippocampal-mediated cognitive behaviors. Before the presentation of the foot-shock, Dp(16)1Yey/+ mice and their wild-type littermates had a similar baseline freezing level in the test (p>0.05; Fig. 3B). Both Dp(16)1Yey/+ mice and the wild-type littermates increased their freezing behavior after being returned to the test chamber 24 hrs after the initial training and foot-shock, but Dp(16)1Yey/+ mice exhibited a decreased freezing level (p<0.005, Fig. 3B). After the 24-hr contextual test, the freezing levels of Dp(16)1Yey/+ and their wild-type littermates in an altered context were analyzed and found to be similar (p>0.05), although they were significantly lower than the freezing levels earlier when the mice were exposed to the original context. This result suggests that neither mouse strain exhibited generalized freezing in all conditions (Balogh et al., 2002). Seventy-two hrs after the initial training, the mice were returned to the original context for the additional test, and Dp(16)1Yey/+ mice again froze less (p<0.05, Fig. 3B). To facilitate interpretation of the above data, we performed a foot-shock test, as described in Materials and Methods, and detected no difference in the mean threshold of the current to elicit flinching or vocalizing between Dp16)1Yey/+ mice and their wild-type littermates (p>0.05) (Fig. 4). Therefore, we can conclude that the Dp(16)1Yey/+ mice were impaired in context-associated learning. The results from our other contextual fear conditioning experiments show that Dp(10)1Yey/+ or Dp(17)1Yey/+ mice performed at a similar proficiency to their wild-type littermates in this test for context-associated learning (Figs. 3A, 3C).
To assess the impact of individual syntenic regions on hippocampal synaptic plasticity at the physiological level, we carried out an in vitro electrophysiological analysis of the CA1 region of the hippocampus in brain slices. We focused on hippocampal LTP, an activity-dependent, sustained increase in the efficacy of synaptic transmission in the hippocampus, which is viewed as an important cellular manifestation of learning and memory (Bear and Abraham, 1996; Bliss and Collingridge, 1993; Malenka and Nicoll, 1999). The field excitatory postsynaptic potentials (fEPSPs) of the CA1 region of the hippocampus in brain slices were induced by theta-burst stimulation (TBS). The baseline fEPSPs as well as evoked potentials after the TBS induction were recorded for each brain slice. A comparative analysis of the time-course revealed that hippocampal LTP in Dp(10)1Yey/+, Dp(16)1Yey/+ and Dp(17)1Yey/+ mice were unchanged (p>0.05), decreased (p<0.05) and increased (p<0.05), respectively (Fig. 5).
Duplications in the mutant mice may alter the expression levels of the genes that are associated with normal synaptic plasticity in the hippocampus. To test such a possibility, we analyzed the following LTP markers using real-time RT-PCR with RNA isolated from the hippocampus of the mice at 4 months of age as the templates: Creb1 (Liu et al., 2008; Paul et al., 2010), Dlg1 (Howard et al., 2010; Nakagawa et al., 2004), Dlg4 (Bracchi-Ricard et al., 2008; Chen et al., 2007), Gria1 (Frey et al., 2009; Mead and Stephens, 2003) and Grin1 (Chen et al., 2009; Kew et al., 2000). Our results show the presence of Dp(16)1Yey led to significant decreases in the RNA levels for Dlg1 and Grin1 (Table 1) and suggests that the alterations of the transcriptional levels of these genes may contribute to the impaired hippocampal LTP in Dp(16)1Yey/+ mice. In the same experiment, we confirmed the elevated expression of App in Dp(16)1Yey/+ mice (Li et al., 2007). However, the expression level of App is not altered in Dp(10)1Yey/+ mice or in Dp(17)1Yey/+ mice, suggesting that there are no genes located in either Hsa21 syntenic region on Mmu10 or Mmu17 that when triplicated will affect the expression level of App.
We have recently described the phenotypic observations of Dp(16)1Yey/+ embryos (Li et al., 2007) and Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+ mice (Yu et al., 2010). In this report, we described the first phenotypic observations of the adult Dp(10)1Yey/+, Dp(16)1Yey/+ and Dp(17)1Yey/+ mice.
For the Hsa21 syntenic region on Mmu10, no related segmental trisomic mice have been reported by any other group. Our results derived from Dp(10)1Yey/+ mice show that the duplication of this region did not cause alteration in cognitive behaviors or in hippocampal LTP.
Dp(16)1Yey/+ mice exhibit the cognitively relevant phenotypes associated with DS: impaired learning/memory and decreased hippocampal LTP, which are also the phenotypes of Ts65Dn mice (Costa and Grybko, 2005; Costa et al., 2008; Escorihuela et al., 1995; Kleschevnikov et al., 2004; Reeves et al., 1995; Sago et al., 2000; Siarey et al., 1997). The phenotypic similarity between Dp(16)1Yey/+ and Ts65Dn mice suggests that the critical genes associated with these phenotypes may reside within the Mrpl39-Zfp295 genomic segment (Fig. 1). Compared to Ts65Dn mice, Dp(16)1Yey/+ mice carry three copies of approximately 16 extra Hsa21 gene orthologs on Mmu16 (http://www.ensembl.org; http://genome.ucsc.edu), suggesting that the triplications of these genes did not significantly alter the cognitively relevant phenotypes between these two models. Ts65Dn mice also carry a trisomic segment for the subcentromeric region of Mmu17 that is not syntenic to any region on Hsa21 (Akeson et al., 2001). Our previous study found that this Mmu17 trisomic segment is larger than 5.8 Mb and contains as many as 19 genes, including Synj2 (Li et al., 2007), whose protein product may contribute to neurological phenotypes in Ts65Dn mice (Li et al., 2007; Malecz et al., 2000; Nemoto et al., 1997; Nemoto et al., 2001; Rusk et al., 2003). However, since Dp(16)1Yey/+ mice exhibit DS-related cognitively relevant phenotypes without triplication of this subcentromeric Mmu17 region, it is unlikely that a gene or genes in this specific Mmu17 region contribute significantly to the cognitively relevant phenotypes in Ts65Dn mice. Potential contributions of several genes located in the Hsa21 syntenic region on Mmu16 to hippocampal-mediated cognitive phenotypes have been proposed. The triplication of the Dyrk1a ortholog alone by BAC transgenics causes impaired cognitive behaviors but enhanced hippocampal LTP in mutant mice (Ahn et al., 2006), suggesting that the triplication of Dyrk1a alone is not sufficient to cause the decreased LTP in Dp(16)1Yey/+ mice. As a potassium channel protein, KCNJ6 regulates neuronal excitability by mediating inhibitory effects of G-protein-coupled receptors for neuromodulators and neurotransmitters, such as adenosine A1 receptors and GABAB receptors (Mark and Herlitze, 2000; Yamada et al., 1998). One study suggests that a null mutation of Kcnj6 may be associated with impairment in the depotentiation of hippocampal LTP (Chung et al., 2009). Therefore, the triplication of Kcnj6 may alter the inhibitory tone of the synapse and may then lead to impaired hippocampal LTP. It has also been proposed that the simultaneous triplications of Dyrk1a and Rcan1 could cause NFAT dysfunction, which in turn could lead to cognitively relevant phenotypes (Arron et al., 2006). Evidence of the contribution of triplications of Olig1 and Olig2 to abnormal brain development has recently been reported (Chakrabarti et al., 2010).
For the Hsa21 syntenic region on Mmu17, we show that triplication of this syntenic region in Dp(17)1Yey/+ mice results in increased hippocampal LTP. Interestingly, increased hippocampal LTP has also been observed in Ts1Yah mice with the triplication of only 12 Hsa21 gene orthologs in the syntenic region (Pereira et al., 2009), indicating that the gene(s) responsible for this phenotype is located within the Abcg1-U2af1 region (Fig. 1) and that triplication of the additional 7 Hsa21 gene orthologs on Mmu17 apparently did not alter the hippocampal LTP-related phenotype.
Although our results show that individual duplication mice exhibit different cognitively relevant phenotypes, the compound mice simultaneously carrying all three duplications show reduced hippocampal LTP and impaired learning/memory (Yu et al., 2010). These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Understanding how individual duplications contribute to the final outcome is a key to unraveling the molecular mechanism underlying DS-associated intellectual disability. One such example is to understand how the gene(s) causing increased hippocampal LTP on Mmu16 interacts directly or indirectly with the genes causing decreased hippocampal LTP on Mmu17 as well as how such an interaction(s) leads to the hippocampal LTP-related phenotype in the compound mutant mice. The results described in this report serve as an important step to achieving this objective.
Patients with Down syndrome exhibit the phenotypes in varying degrees, including intellectual disability with ranges extending from severely impaired to low-normal intelligence (Chapman and Hesketh, 2000; Pulsifer, 1996). The genetic basis for such heterogeneity in the severity of phenotypes is unknown, and advances in this area may lead to new strategies for rational development of effective treatments for phenotypic abnormalities, such as intellectual disability in DS. Mouse models could serve as essential genetic reagents for such an effort. However, while Tc1 mice are maintained in a C57BL/6J × 129S8F1 background, Ts65Dn mice are maintained by crossing Ts65Dn females to C57BL/6JEi × 3CH/HeSnjF1 males. The backcrossing of Ts65Dn mice to any inbred strains inevitably led to infertility (Pritchard et al., 2008). This is probably caused by the arrest of gametogenesis due to the presence of the derivative of chromosomal translocation (Burgoyne et al., 1985; de Boer et al., 1986). The backcrossing of Tc1 mice to an inbred strain led to loss of transmission of the human chromosome 21 fragment to the progeny (O'Doherty et al., 2005). Dp(10)1Yey, Dp(16)1Yey, or Dp(17)1Yey as a duplication embedded on a mouse chromosome, does not lead to these problems and can be backcrossed to C57BL/6J mice to generate fertile progeny. Therefore, Dp(10)1Yey/+, Dp(16)1Yey/+ and Dp(17)1Yey/+ mice from different strain backgrounds should facilitate future efforts to identify genetic modifiers for phenotypes in Down syndrome.
Mouse models have contributed significantly to our understanding of DS. The establishment of Dp(10)1Yey/+, Dp(16)1Yey/+ and Dp(17)1Yey/+ mouse models opened up new investigative avenues, and the further characterization of these models will undoubtedly expand our knowledge of DS-associated intellectual disability.
The mutant mice were first established in a 129SvEvxC57BL/6JF1 strain background and were then backcrossed to C57BL/6J mice for five generations. The mutant mice and their littermates were maintained at a temperature- and humidity-controlled animal facility. All mice used in the experiments were 2-4 months old. We used Dp(10)1Yey/+ mice (n=14), the wild-type littermates of Dp(10)1Yey/+ (n=15), Dp(16)1Yey/+ mice (n=15), the wild-type littermates of Dp(16)1Yey/+ (n=14), Dp(17)1Yey/+ mice (n=13) and the wild-type littermates of Dp(17)1Yey/+ (n=15) for behavioral analysis. Before behavioral experiments, each mouse was pre-handled for two minutes every day for a week. The experimental procedures were approved by the Institutional Animal Care and Use Committee.
Real-time quantitative PCR was used to analyze RNA levels of the genes that are associated with normal synaptic plasticity in the hippocampus. Gapdh is located on Mmu 6 and served as a reference gene for all the mice examined. Total RNAs were isolated from mouse hippocampi using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA). 1 μg of the pooled RNA from three mice with the same genotype was used to generate cDNA by using Superscript version III reverse transcriptase (Invitrogen Corp., Carlsbad, CA). The specific primers and probes for the genes were obtained from the TagMan® Gene Expression Assays System of Applied Biosystems, Inc. A 0.5 μg of cDNA from each genotype was analyzed by ABI 7900HT Real-Time Thermocycler (Applied Biosystems, Foster City, CA) with the following amplification conditions: an initial activation and denaturation at 95° C for 10 min, followed by 40 cycles of denaturation at 95° C for 15 sec and primer annealing and extension at 60° C for 1 min.
Morris water maze tests were performed in a circular pool (152 cm in diameter) and based on an established protocol used in one of our laboratories (Clapcote and Roder, 2004; Clapcote et al., 2005). In the tests, each mouse was released into the water at one of the cardinal compass points (north, south, east or west) and allowed the time to find a platform. After each mouse in the testing squad had completed trial 1, the next trial was begun. The order of release points was predetermined to be pseudorandom but not sequential. We recorded and analyzed the experimental data using HVS Water 2020, an imaging-tracking and analysis system (HVS Image Ltd.). The amount of time spent finding the platform (latency), the distance traveled (path-length) and swimming speed were recorded. Each mouse had four trials each day in visible-platform and hidden-platform training trials, which were carried out on day 1 and days 2-7, respectively. On day 8, a probe test was performed in which the platform was removed from the water and each mouse was allowed 60 sec to search the pool. The time spent in each quadrant was recorded.
The contextual fear conditioning test was carried out according to the procedures described by our laboratories (Clapcote et al., 2005; Lu et al., 1997). The test chamber floor was a grid of stainless steel rods connected to an electric shock generator. A video camera was mounted on the front wall and a ceiling light illuminated the chamber interior through the transparent ceiling. Prior to conditioning, each mouse had 2 min to explore the test chamber, and this baseline activity was monitored by the Fear Conditioning Video Tracking System (Med Associates Inc., St. Albans, VT). A foot-shock (1 mA scrambled) was then administered for 2 sec, which was controlled by a computer program (Video Freeze Software V.1.8, Med Associates Inc.). Each mouse was returned to the test chamber after approximately 24 hrs and monitored for freezing behavior for 3 min, during which no foot-shock was delivered. Two hrs after the 24-hr context test, the context was changed by cleaning the chamber with 1% acetic acid, covering the grid floor with a sheet of Perspex and inserting two sheets of Perspex into the chamber to give it a prism shape. Each mouse was placed into the altered context and allowed 2 min for exploration. Freezing behavior in the altered context was used to measure generalized freezing, i.e., simply suppressed activity during all conditions (Balogh et al., 2002). Seventy-two hrs after the initial training, each mouse was again placed in the original test chamber for a final 3 min of observation without any foot-shock being delivered.
The fear conditioning test chamber was used to perform a foot-shock sensitivity test. A foot-shock was delivered every 10 sec, starting at 0.05 mA with a 0.05 mA increment between each shock (Rosa et al., 2007). The minimal level of current needed to elicit flinching or vocalizing was recorded.
The measurement of hippocampal LTP was carried out as described (Jia et al., 1996; Meng et al., 2002). Briefly, hippocampal slices (400 μm) from the mice were prepared as described (Henderson et al., 2001), placed in the temperature-controlled recording chamber and superfused with oxygenated artificial cerebrospinal fluid, which contains 120 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 26 mM NaHCO3, 2.5 mM CaCl2, and 11 mM D-glucose. To record fEPSPs, the stimulating and recording electrodes were placed in the Schaffer collaterals and stratum radiatum of CA1, respectively, separated by a distance of 200-300 μm. LTP was induced by TBS (15 bursts of 4 pulses at 100 Hz, delivered at an interburst interval of 200 ms) through the stimulating electrode. We performed electrophysiological recordings for 80 min for each brain slice, including 20 min of baseline recording and 60 min after the TBS induction. pClamp9.2 and the Clampfit 9.2 program (Molecular Devices, Sunnyvale, CA) were used to obtain and analyze the traces.
One-way ANOVA was used to compare the data from Morris water maze probe tests, contextual fear conditioning tests and foot-shock sensitivity tests between genotypes. No effects from gender were detected by ANOVA in all the behavioral tests and, thus, the data from both genders were pooled and analyzed together for these experiments. Data from the six-day training trials of the Morris water maze hidden-platform version were analyzed using a two-way (genotype × day) ANOVA with the genotype as a between-subjects factor and the day as a repeated-measures factor. The electrophysiology data were analyzed with a Student's t test. All values reported in the text and figures are presented as means ± S.E.M.
This project was supported in part by grants from the Roswell Park Alliance Foundation, the Louis Sklarow Memorial Fund, the Fondation Jerome Lejeune, the Children's Guild Foundation and the NIH (R01HL091519 and R01NS066072) and by the Cancer Center Support Grant from NIH (P30CA016056).
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