Altered intrinsic and network properties of neocortical neurons in the Ts65Dn mouse model of Down syndrome.
ABSTRACT: All individuals with Down syndrome (DS) have a varying but significant degree of cognitive disability. Although hippocampal deficits clearly play an important role, behavioral studies also suggest that deficits within the neocortex contribute to somatosensory deficits and impaired cognition in DS. Using thalamocortical slices from the Ts65Dn mouse model of DS, we investigated the intrinsic and network properties of regular spiking neurons within layer 4 of the somatosensory cortex. In these neurons, the membrane capacitance was increased and specific membrane resistance decreased in slices from Ts65Dn mice. Examination of combined active and passive membrane properties suggests that trisomic layer 4 neurons are less excitable than those from euploid mice. The frequencies of excitatory and inhibitory spontaneous synaptic activities were also reduced in Ts65Dn neurons. With respect to network activity, spontaneous network oscillations (Up states) were shorter and less numerous in the neocortex from Ts65Dn mice when compared to euploid. Up states evoked by electrical stimulation of the ventrobasal nucleus (VBN) of the thalamus were similarly affected in Ts65Dn mice. Additionally, monosynaptic EPSCs and polysynaptic IPSCs evoked by VBN stimulation were significantly delayed in layer 4 regular spiking neurons from Ts65Dn mice. These results indicate that, in the Ts65Dn model of DS, the overall electrophysiological properties of neocortical neurons are altered leading to aberrant network activity within the neocortex. Similar changes in DS individuals may contribute to sensory and cognitive dysfunction and therefore may implicate new targets for cognitive therapies in this developmental disorder.
Project description:Down syndrome (DS), the most frequent genetic cause of intellectual disability and developmental delay, results from impaired neural stem cell proliferation and differentiation. Impaired neurogenesis in the neocortex, hippocampus and cerebellum is believed to be the underlying cause of learning and behavioral deficits in the Ts65Dn mouse model of DS. Aggressive sensorimotor and cognitive therapies have shown promise in mitigating the cognitive disabilities in DS but these behavioral therapies have not yet been investigated at the cellular level. Here, using the Ts65Dn mouse model of DS, we demonstrate that a combination of environmental enrichment and physical exercise starting in juvenile mice (postnatal day 18) markedly increases cell proliferation, neurogenesis and gliogenesis in the hippocampal dentate gyrus (DG) and the forebrain subventricular zone (SVZ) of both male and female mice. Enrichment and exercise increased the rate of Ts65Dn DG neurogenesis to be comparable to that of the nonenriched euploid group, while the effect on SVZ neurogenesis was reduced and seen only after prolonged exposure. These results clearly indicate that in a comprehensive stimulatory environment, the postnatal DS brain has the intrinsic capability of improving neurogenesis and gliogenesis to the levels of normal matched controls and that this cellular response underlies the cognitive improvement seen following behavioral therapies.
Project description:Down syndrome (DS), caused by trisomy of human chromosome 21 (chr21), is the most common genetic cause of intellectual disability. The Ts65Dn mouse model of DS is trisomic for orthologs of 94 chr21-encoded, confirmed protein-coding genes and displays a number of behavioral deficits. Recently, Ts65Dn mice were shown to be hypersensitive to the locomotor stimulatory effects of the high-affinity N-methyl-d-aspartate (NMDA) receptor (NMDAR) channel blocker, MK-801. This is consistent with the functions of several chr21 proteins that are predicted directly or indirectly to impact NMDAR function or NMDAR-mediated signaling. In this study, we show that a second mouse model of DS, the Ts1Cje, which is trisomic for 70 protein-coding genes, is also hypersensitive to MK-801. To investigate the molecular basis for the responses to MK-801, we have measured levels of a subset of chr21 and phosphorylated non-chr21 proteins, in the cortex and hippocampus of Ts65Dn and Ts1Cje mice and euploid controls, with and without treatment with MK-801. We show that in euploid mice, the chr21-encoded proteins, TIAM1 and DYRK1A, and phosphorylation of AKT, ERK1/2 and the transcription factor ELK are involved in the MK-801 response. However, in both Ts65Dn and Ts1Cje mice, levels of phosphorylation are constitutively elevated in naïve, unstimulated mice, and the MK-801-induced changes in TIAM1 and DYRK1A and in phosphorylation are either absent or abnormal, with both genotype and brain-region-specific patterns. These results emphasize the complexities of the pathway perturbations that arise with segmental trisomy.
Project description:Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.
Project description:BACKGROUND:Down syndrome (DS), caused by the triplication of chromosome 21, results in a constellation of clinical features including changes in intellectual and motor function. Although altered neural development and function have been well described in people with DS, few studies have investigated the etiology underlying the observed motor phenotypes. Here, we examine the development, patterning, and organization of the spinal cord throughout life in the Ts65Dn mouse, a model that recapitulates many of the motor changes observed in people with DS. METHODS:Spinal cords from embryonic to adult animals were processed for gene and protein expression (immunofluorescence) to track the spatiotemporal development of excitatory and inhibitory neurons and oligodendroglia. Postnatal analyses were focused on the lumbar region due to the reflex and gait abnormalities found in Ts65Dn mice and locomotive alterations seen in people with DS. RESULTS:Between embryonic days E10.5 and E14.5, we found a larger motor neuron progenitor domain in Ts65Dn animals containing more OLIG2-expressing progenitor cells. These disturbed progenitors are delayed in motor neuron production but eventually generate a large number of ISL1+ migrating motor neurons. We found that higher numbers of PAX6+ and NKX2.2+ interneurons (INs) are also produced during this time frame. In the adult lumbar spinal cord, we found an increased level of Hb9 and a decreased level of Irx3 gene expression in trisomic animals. This was accompanied by an increase in Calretinin+ INs, but no changes in other neuronal populations. In aged Ts65Dn animals, both Calbindin+ and ChAT+ neurons were decreased compared to euploid controls. Additionally, in the dorsal corticospinal white matter tract, there were significantly fewer CC1+ mature OLs in 30- and 60-day old trisomic animals and this normalized to euploid levels at 10-11?months. In contrast, the mature OL population was increased in the lateral funiculus, an ascending white matter tract carrying sensory information. In 30-day old animals, we also found a decrease in the number of nodes of Ranvier in both tracts. This decrease normalized both in 60-day old and aged animals. CONCLUSIONS:We show marked changes in both spinal white matter and neuronal composition that change regionally over the life span. In the embryonic Ts65Dn spinal cord, we observe alterations in motor neuron production and migration. In the adult spinal cord, we observe changes in oligodendrocyte maturation and motor neuron loss, the latter of which has also been observed in human spinal cord tissue samples. This work uncovers multiple cellular perturbations during Ts65Dn development and aging, many of which may underlie the motor deficits found in DS.
Project description:Down syndrome (DS) is a high-incidence genetic pathology characterized by severe impairment of cognitive functions, including declarative memory. Impairment of hippocampus-dependent long-term memory in DS appears to be related to anatomo-functional alterations of the hippocampal trisynaptic circuit formed by the dentate gyrus (DG) granule cells - CA3 pyramidal neurons - CA1 pyramidal neurons. No therapies exist to improve cognitive disability in individuals with DS. In previous studies we demonstrated that pharmacotherapy with fluoxetine restores neurogenesis, granule cell number and dendritic morphology in the DG of the Ts65Dn mouse model of DS. The goal of the current study was to establish whether treatment rescues the impairment of synaptic connectivity between the DG and CA3 that characterizes the trisomic condition. Euploid and Ts65Dn mice were treated with fluoxetine during the first two postnatal weeks and examined 45-60 days after treatment cessation. Untreated Ts65Dn mice had a hypotrophyc mossy fiber bundle, fewer synaptic contacts, fewer glutamatergic contacts, and fewer dendritic spines in the stratum lucidum of CA3, the terminal field of the granule cell projections. Electrophysiological recordings from CA3 pyramidal neurons showed that in Ts65Dn mice the frequency of both mEPSCs and mIPSCs was reduced, indicating an overall impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons. In treated Ts65Dn mice all these aberrant features were fully normalized, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The positive effects of fluoxetine on the DG-CA3 system suggest that early treatment with this drug could be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions.
Project description:INTRODUCTION:The Ts65Dn mouse is the most widely used animal model of Down syndrome (DS). Differences in autonomic regulation of heart rate variability (HRV) in individuals with DS have been hypothesized. Pharmacological studies in animal models may help us understand mechanisms underlying observed changes in HRV in people with DS. OBJECTIVE:To investigate the use a new, noninvasive technique to assess cardiac autonomic modulation in Ts65Dn mice under the effect of adrenergic and cholinergic agonists. METHOD:We recorded electrocardiograms (ECGs) from 12 Ts65Dn and 12 euploid control mice. A 30-min baseline recording was followed by the injection of an adrenergic (isoproterenol [Iso]) or cholinergic (carbachol [CCh]) agonist. Heart rate and HRV were analyzed using a series of methods customized for mice. RESULTS AND DISCUSSION:The ECG apparatus described here allowed us to detect noninvasively long series of heartbeats in freely-moving animals. During baseline conditions, the yield of detectable heartbeats was 3%-27% of the estimated total number of events, which increased to 35%-70% during the 15-min period after either Iso or CCh injections. Ts65Dn mice displayed a robust enhanced Iso-induced negative chronotropic rebound response compared with euploid control mice. We observed a significantly smaller CCh response in Ts65Dn versus control euploid mice in the 6- to 10-min-interval postcarbachol injection. CONCLUSION:This work showed that the techniques described here are sufficient for this type of study. However, future studies involving the use of more selective pharmacological agents and/or genetic manipulations will be key to advance a mechanistic understanding of cardiac autonomic regulation in DS.
Project description:Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca(2+)]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in ?f / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype.
Project description:Down syndrome (DS), a genetic condition leading to intellectual disability, is characterized by triplication of human chromosome 21. Neuropathological hallmarks of DS include abnormal central nervous system development that manifests during gestation and extends throughout life. As a result, newborns and adults with DS exhibit cognitive and motor deficits and fail to meet typical developmental and lack independent life skills. A critical outstanding question is how DS-specific prenatal and postnatal phenotypes are recapitulated in different mouse models. To begin answering this question, we developed a life span approach to directly compare differences in embryonic brain development, cellularity, gene expression, neonatal and adult behavior behavior in three cytogenetically distinct mouse models of DS—Ts1Cje, Ts65Dn and Dp16/1Yey (Dp16). In the last two decades multiple therapeutic trials have been attempted to improve cognition in humans with DS but the results of these interventions lacked efficacy despide their succes in the Ts65Dn mouse model of DS. To better understand how phenotypic changes in humans in DS are recapitulated in different mouse models, we copared embryonic brain development and gene expression, perinatal behavior and brain excitatoty/inhibitory cell distribution, and adult behavior and gene expression in the Dp16, Ts65Dn and Ts1Cje mouse models of DS. The objectives of this study were to determine the best model(s) for prenatal and postnatal therapeutic trials and to identify treatment endpoints that can be used to evaluate the fficacy of these therapies prior to human clinical trials. Our data showed that, at embryonic day 15.5, Ts65Dn mice are the most profoundly and consistently affected with respect to somatic growth, brain morphogenesis, and neurogenesis compared to Ts1Cje and Dp16 embryos. However, gene expression results show that both Ts65Dn and Ts1Cje embryonic forebrains have a relatively high number of differentially expressed genes compared to Dp16, with little overlap in gene identities and genomic distribution observed among these models. Additionally, postnatal histological analyses show varying degrees of cell population and brain histogenesis abnormalities among the three strains. Behavioral testing also highlights differences among the models in their ability to meet various developmental milestones. At adulthood, Ts65Dn and Ts1Cje showed hyperactive behavior in the open field test but not Dp16 mice. In the fear conditioning test, all three strains showed lower freezing versus eupploid mice; Dp16 were the most severely affected. In the Morris water maze, Ts65Dn showed significant delays in the hidden platform, probe and reversal trials. Dp16 mice showed milder deficits in the hidden platform trial but severe deficits in reversal. Ts1Cje mice had no spatial memory deficits. In the rotarod test, Dp16 performed poorly in fixed and accelerating speed trials, while Ts1Cje was only abnormal at high speeds. Ts65Dn rotarod performance was unaffected. Compared to euploid, Ts65Dn had a higher number of differentially expressed (DEX) genes in cortex and cerebellum, while Ts1Cje had more DEX genes in hippocampus and cerebellum. Dp16 had the lowest number of DEX genes in all regions analyzed. Pathway analyses highlighted commonly dysregulated pathways, including G-protein signaling, oxidative stress, interferon signaling, glycosylation and disulfide bonds. Overall design: At embryonic day 15.5 (E15.5), we analyzed forebrain gene expression in Dp16 (n=6) and euploid littermates (n=6), Ts65Dn (n=5) and euploid littermates (n=6), and Ts1Cje (n=5) and euploid littermates (n=5) using Affymetrix mouse gene 1.0 ST array. At adulthood, we investigated gene expression and pathway abnormalities in the cerebral cortex, hippocampus and cerebellum obtained from 6-7 month old Dp16 (n=5) and euploid littermates (n=5), Ts65Dn (n=5) and euploid littermates (n=5), and Ts1Cje (n=5) and euploid littermates (n=5) using the same Affymetrix arrays. Data were normalized and analyzed to identify and accurately map genes that are significantly differentially expressed. Functional analyses were performed using DAVID and Ingenuity Pathway Analysis (IPA) to better characterize pathway similarities and differences in these mouse models. In separate experiments, we used histology, neonatal and adult behavioral approaches to link gene exression differences to potential cellular and behavioral deficits in each mouse model.
Project description:Down syndrome is frequently associated with complex difficulties in oromotor development, feeding, and swallowing. However, the muscle phenotypes underlying these deficits are unclear. We tested the hypotheses that the Ts65Dn mouse model of DS has significantly altered myosin heavy chain (MyHC) isoform profiles of the muscles involved in feeding and swallowing, as well as reductions in the speed of these movements during behavioral assays. SDS-PAGE, immunofluorescence, and qRT-PCR were used to assess MyHC isoform expression in pertinent muscles, and functional feeding and swallowing performance were quantified through videofluoroscopy and mastication assays. We found that both the anterior digastric (ADG) and posterior digastric (PDG) muscles in 11-day old and 5-6 week old Ts65Dn groups showed significantly lower MyHC 2b protein levels than in age-matched euploid control groups. In videofluoroscopic and videotape assays used to quantify swallowing and mastication performance, 5-6 week old Ts65Dn and euploid controls showed similar swallow rates, inter-swallow intervals, and mastication rates. In analysis of adults, 10-11 week old Ts65Dn mice revealed significantly less MyHC 2b mRNA expression in the posterior digastric, but not the anterior digastric muscle as compared with euploid controls. Analysis of MyHC 2b protein levels across an adult age range (10-53 weeks of age) revealed lower levels of MyHC 2b protein in the PDG of Ts65Dn than in euploids, but similar levels of MyHC 2b in the ADG. Cumulatively, these results indicate biochemical differences in some, but not all, muscles involved in swallowing and jaw movement in Ts65Dn mice that manifest early in post-natal development, and persist into adulthood. These findings suggest potential utility of this model for future investigations of the mechanisms of oromotor difficulties associated with Down syndrome.
Project description:Decrease of GABAergic transmission has been proposed to improve memory functions. Indeed, inverse agonists selective for ?5 GABA-A-benzodiazepine receptors (?5IA) have promnesiant activity. Interestingly, we have recently shown that ?5IA can rescue cognitive deficits in Ts65Dn mice, a Down syndrome mouse model with altered GABAergic transmission. Here, we studied the impact of chronic treatment with ?5IA on gene expression in the hippocampus of Ts65Dn and control euploid mice after being trained in the Morris water maze task. In euploid mice, chronic treatment with ?5IA increased IEGs expression, particularly of c-Fos and Arc genes. In Ts65Dn mice, deficits of IEGs activation were completely rescued after treatment with ?5IA. In addition, normalization of Sod1 overexpression in Ts65Dn mice after ?5IA treatment was observed. IEG expression regulation after ?5IA treatment following behavioral stimulation could be a contributing factor for both the general promnesiant activity of ?5IA and its rescuing effect in Ts65Dn mice alongside signaling cascades that are critical for memory consolidation and cognition.