Synaptic structural abnormalities in the Ts65Dn mouse model of Down Syndrome

The Ts65Dn mouse is a genetic model for Down syndrome. Although this mouse shows abnormalities in cognitive function that implicate hippocampus as well as marked deficits in hippocampal long-term potentiation, the structure of the hippocampus has been little studied. We characterized synaptic structure in Ts65Dn and control (2N) mice, studying the hippocampus (fascia dentata, CA1) as well as the motor and somatosensory cortex, entorhinal cortex, and medial septum. Confocal microscopy was used to examine immunostained presynaptic boutons and to detail the structure of dendrites after Lucifer yellow microinjection. Both presynaptic and postsynaptic elements were significantly enlarged in Ts65Dn in all regions examined. The changes were detected at the youngest age examined (postnatal day 21) and in adults. In studies detailing the changes in fascia dentata and motor cortex, the enlargement of spines affected the entire population, resulting in the presence of spines whose volume was greatly increased. Electron microscopy confirmed that boutons and spines were enlarged and demonstrated abnormalities in the internal membranes of both. In addition, spine density was decreased on the dendrites of dentate granule cells, and there was reorganization of inhibitory inputs, with a relative decrease in inputs to dendrite shafts and an increase in inputs to the necks of spines. Taken together, the findings document widespread abnormalities of synaptic structure that recapitulate important features seen in Down syndrome. They establish the Ts65Dn mouse as a model for abnormal synapse structure and function in Down syndrome and point to the importance of studies to elucidate the mechanisms responsible for synapse enlargement.     

Fluoxetine rescues deficient neurogenesis in hippocampus of the Ts65Dn mouse model for Down syndrome

The Ts65Dn mouse, an adult model of Down syndrome displays behavioral deficits consistent with a dysfunctional hippocampus, similar to that seen with DS. In looking for mechanisms underlying these performance deficits, we have assessed adult neurogenesis in the dentate gyrus of Ts65Dn. Under untreated conditions, Ts65Dn mice (2-5 months old) showed markedly fewer BrdU-labeled cells than euploid animals. Chronic antidepressant treatment for over 3 weeks with the serotonin selective reuptake inhibitor, fluoxetine, increased neurogenesis in the Ts65Dn to comparable levels seen in the euploid by augmenting both proliferation and survival of BrdU-labeled cells in the subgranular layer and granule cell layer of the hippocampus, respectively.     

Excitatory‐inhibitory relationship in the fascia dentata in the Ts65Dn mouse model of down syndrome

Down syndrome (DS) is a neurological disorder causing impaired learning and memory. Partial trisomy 16 mice (Ts65Dn) are a genetic model for DS. Previously, we demonstrated widespread alterations of pre‐ and postsynaptic elements and physiological abnormalities in Ts65Dn mice. The average diameter of presynaptic boutons and spines in the neocortex and hippocampus was enlarged. Failed induction of long‐term potentiation (LTP) due to excessive inhibition was observed. In this paper we investigate the morphological substrate for excessive inhibition in Ts65Dn. We used electron microscopy (EM) to characterize synapses, confocal microscopy to analyze colocalization of the general marker for synaptic vesicle protein with specific protein markers for inhibitory and excitatory synapses, and densitometry to characterize the distribution of the receptor and several proteins essential for synaptic clustering of neurotransmitter receptors. EM analysis of synapses in the Ts65Dn vs. 2N showed that synaptic opposition lengths were significantly greater for symmetric synapses (～18%), but not for asymmetric ones. Overall, a significant increase in colocalization coefficients of glutamic acid decarboxylase (GAD)65/p38 immunoreactivity (IR) (～27%) and vesicular GABA transporter (VGAT)/p38 IR (～41%) was found, but not in vesicular glutamate transporter 1 (VGLUT1)/p38 IR. A significant overall decrease of IR in the hippocampus of Ts65Dn mice compared with 2N mice for glutamate receptor 2 (GluR2; ～13%) and anti‐γ‐aminobutyric acid (GABA)_A receptor β2/3 subunit (～20%) was also found. The study of proteins essential for synaptic clustering of receptors revealed a significant increase in puncta size for neuroligin 2 (～13%) and GABA_A receptor‐associated protein (GABARAP; ～13%), but not for neuroligin 1 and gephyrin. The results demonstrate a significant alteration of inhibitory synapses in the fascia dentata of Ts65Dn mice. J. Comp. Neurol. 512:453–466, 2009. © 2008 Wiley‐Liss, Inc.     

Synaptic deficit in the temporal cortex of partial trisomy 16 (Ts65Dn) mice

Down syndrome results from triplication of human chromosome 21. The distal end of mouse chromosome 16 shares a large region of genetic homology with the Down syndrome ‘critical region' of human chromosome 21. Therefore, a partially trisomic mouse (Ts65Dn) that possesses a triplication of the distal region of chromosome 16 has been developed as a putative model for Down syndrome. Ts65Dn mice display learning and memory deficits. However, despite the importance of preserved synaptic integrity for learning and memory, the ultrastructure of neural connectivity has not yet been studied in Ts65Dn mice. Therefore, the density and apposition zone length of synapses in the temporal cortex of aged Ts65Dn mice (n=4) were compared with those in diploid controls (n=4), using quantitative electron microscopy. There were significantly less (30%) asymmetric synapses in the temporal cortex of Ts65Dn mice than in controls (t=−5.067; p=0.023). However, there was no significant difference between the mean density of symmetric synapses in Ts65Dn mice and control mice. In addition, the mean synaptic apposition lengths of both asymmetric (15%; t=9.812, p<0.0001) and symmetric (11%; t=5.582; p<0.0001) synapses were significantly larger in Ts65Dn mice than in controls. These results suggest that excitatory synapses are preferentially affected in Ts65Dn mice and that there is an attempt to compensate for the deficit of asymmetric synapses by increasing the contact zone area of existing synapses. The results may also reveal the morphological basis for the learning and memory deficits observed in Ts65Dn mice and have a bearing on the cognitive deficits in Down syndrome in old age.     

Three-dimensional synaptic ultrastructure in the dentate gyrus and hippocampal area CA3 in the Ts65Dn mouse model of Down syndrome

Down syndrome (DS) results from trisomy of human chromosome 21. Ts65Dn mice are an established model for DS and show several phenotypes similar to those in people with DS. However, there is little data on the structural plasticity of synapses in the trisynaptic pathway in the hippocampus. Here we investigate 3D ultrastructure of synapses in the hippocampus of age-matched control (2N) and Ts65Dn male mice. Serial ultrathin sections and 3D reconstructions characterize synapses in the middle molecular layer (MML) of dentate gyrus and in thorny excrescences (TEs) in proximal portions of apical dendrites of CA3 pyramidal neurons. 3D analysis of synapses shows phenotypes that distinguish Ts65Dn from 2N mice. For the MML, synapse density was reduced by 15% in Ts65Dn vs. 2N mice (P < 0.05). Comparative 3D analyses demonstrate a significant decrease in the number of thorns per TE in CA3 in Ts65Dn vs. 2N mice (by ≈45%, P = 0.01). Individual thorn volume was 3 times smaller in Ts65Dn vs. 2N mice (P = 0.02). A significant decrease in the number of thorn projections per TE in Ts65Dn vs. 2N mice was accompanied by a decrease of filopodium-like protrusions on the surface of TEs (P = 0.02). However, the volume of postsynaptic densities in CA3 Ts65Dn and 2N mice was unchanged (P = 0.78). Our findings suggest that the high degree of plasticity of CA3 thorns may be connected with their filopodial origin. Alterations of 3D synaptic structure in Ts65Dn mice may further contribute to the diminished plasticity in DS.     

The Ts65Dn mouse model of Down syndrome shows reduced expression of the Bcl-XL antiapoptotic protein in the hippocampus not accompanied by changes in molecular or cellular markers of cell death

The Ts65Dn (TS) mouse, the most widely used model of Down syndrome (DS), has a partial trisomy of a segment of chromosome 16 that is homologous to the distal part of human chromosome 21. This mouse shares many phenotypic characteristics with people with DS including neuromorphological, neurochemical, and cognitive disturbances. Both TS and DS brains show earlier aging and neurodegeneration. Since fibroblast cultures from TS mice and human DS hippocampal regions show increased apoptotic cell death it has been suggested that alterations in cerebral apoptosis might be implicated in the cognitive deficits found in TS mice and in people with DS. In the present study we have evaluated brain expression levels of several proapoptotic and antiapoptotic proteins from the mitochondrial (Bcl-2, Bcl-X_L, Bax and Bad) and the extrinsic (Fas-R and Fas-L) apoptotic pathways as well as the final executioner caspase-3, in the cortex and hippocampus of TS mice. No significant alterations in the expression levels of the proapoptotic Bad and Bax or the antiapoptotic Bcl-2 proteins in the cortex or hippocampus were found in TS mice. However, TS mice showed downregulation of Bcl-X_L in the hippocampus. In the extrinsic pathway we found unchanged levels of Fas-L in both structures and also in the expression levels of Fas-R in the hippocampus. Although Bcl-X_L downregulation suggests that the hippocampus of TS mice is less protected against programmed cell death, we did not find any evidence for increased apoptosis in TS mice since neither TUNEL-positive cells nor active caspase-3 expression were found in cortex or hippocampus of TS or CO mice.     

GABAergic hyperinnervation of dentate granule cells in the Ts65Dn mouse model of down syndrome: Exploring the role of App

It has been suggested that increased GABAergic innervation in the hippocampus plays a significant role in cognitive dysfunction in Down syndrome (DS). Bolstering this notion, are studies linking hyper‐innervation of the dentate gyrus (DG) by GABAergic terminals to failure in LTP induction in the Ts65Dn mouse model of DS. Here, we used extensive morphometrical methods to assess the status of GABAergic interneurons in the DG of young and old Ts65Dn mice and their 2N controls. We detected an age‐dependent increase in GABAergic innervation of dentate granule cells (DGCs) in Ts65Dn mice. The primary source of GABAergic terminals to DGCs somata is basket cells (BCs). For this reason, we assessed the status of these cells and found a significant increase in the number of BCs in Ts65Dn mice compared with controls. Then we aimed to identify the gene/s whose overexpression could be linked to increased number of BCs in Ts65Dn and found that deleting the third copy of App gene in Ts65Dn mice led to normalization of the number of BCs in these mice. Our data suggest that App overexpression plays a major role in the pathophysiology of GABAergic hyperinnervation of the DG in Ts65Dn mice. © 2016 Wiley Periodicals, Inc.     

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

Down syndrome (DS), the leading genetic cause of mental retardation, is characterized by reduced number of cortical neurons and brain size. The occurrence of these defects starting from early life stages points at altered developmental neurogenesis as their major determinant. The goal of our study was to obtain comparative evidence for impaired neurogenesis in the hippocampal dentate gyrus (DG) of DS fetuses and Ts65Dn mice, an animal model for DS. Cell proliferation in human fetuses was evaluated with Ki-67 (a marker of cells in S + G(2) + M phases of cell cycle) and cyclin A (a marker of cells in S phase) immunohistochemistry. We found that in the DG of DS fetuses the number of proliferating cells was notably reduced when compared with controls. A similar reduction was observed in the germinal matrix of the lateral ventricle. In both structures, DS fetuses showed a reduced ratio between cyclin A- and Ki-67-positive cells when compared with controls, indicating that they had a reduced number of cycling cells in S phase. In the DG of P2 Ts65Dn mice cell proliferation, assessed 2 h after an injection of bromodeoxyuridine (BrdU), was notably reduced, similarly to DS fetuses. After 28 days, Ts65Dn mice had still less BrdU-positive cells than controls. Phenotypic analysis of the surviving cells showed that Ts65Dn mice had a percent number of cells with astrocytic phenotype larger than controls. Using phospho-histone H3 immunohistochemistry we found that both DS fetuses and P2 Ts65Dn mice had a higher number of proliferating cells in G(2) and a smaller number of cells in M phase of cell cycle. Results provide novel evidence for proliferation impairment in the hippocampal DG of the DS fetal brain, comparable to that of the P2 mouse model, and suggest that cell cycle alterations may be critical determinants of the reduced proliferation potency.     

Alteration of NO-producing system in the basal forebrain and hypothalamus of Ts65Dn mice: an immunohistochemical and histochemical study of a murine model for Down syndrome

Ts65Dn mice have been developed as a model for Down syndrome (DS). Because of its involvement in complex behaviors, including sexual and aggressive behaviors, we investigated the nitric oxide (NO) system in specific brain regions of these mutant mice (TS) after isolation-induced aggression. Male TS mice displayed significantly higher aggression than wild type (WT) mice and the comparison of the NO system, both with immunohistochemical and histochemical methods, resulted in robust differences between TS and WT mice in the hypothalamic paraventricular nucleus, in the nucleus of the diagonal band and in the medial septum, but not in the striatum of TS mice. In conclusion, we document alterations in the neuronal NO system of the TS mouse model of DS, suggesting a correlation of the behavioral aggressiveness with deficient NO production.     

Hippocampal hypocellularity in the Ts65Dn mouse originates early in development

Ts65Dn, a well-characterized animal model for Down syndrome, has three copies of the distal end of mouse chromosome 16 and therefore has segmental trisomy for orthologs for nearly half of the genes located on human chromosome 21. Ts65Dn mice have learning and memory impairments, especially in tasks involving the hippocampus. Previous studies have shown that older adult Ts65Dn mice have structural abnormalities in the hippocampus including fewer granule cells in dentate gyrus and more pyramidal cells in the CA3 subfield of cornus ammonis. However, it is not clear whether those changes are secondary to the age-related neurodegeneration of the basal forebrain cholinergic neurons that project to the hippocampus or if they originate earlier during hippocampal development. To address this question, we performed a quantitative study of the hippocampal volume and the numbers of granule cell and pyramidal neurons in young (postnatal day 6, P6) and adult (3-month-old) mice using the optical fractionator method. At P6, Ts65Dn mice had 20% fewer granule cells in dentate gyrus than did euploid littermates. Similarly, compared to euploid, P6 trisomic mice showed an 18% reduction in mitotic cells in the granule cell layer and the hilus, where granule cell precursors divide to generate the internal granule cell layer. Granule cell hypocellularity persists in 3-month-old Ts65Dn mice before the onset of cholinergic atrophy. The hypocellularity seen in the trisomic adult hippocampus originates early in development and may contribute to specific cognitive deficits in these mice.     

Spatial Learning Drives Rapid Goal Representation in Hippocampal Ripples without Place Field Accumulation or Goal-Oriented Theta Sequences

The hippocampus is critical for rapid acquisition of many forms of memory, although the circuit-level mechanisms through which the hippocampus rapidly consolidates novel information are unknown. Here, the activity of large ensembles of hippocampal neurons in adult male Long-Evans rats was monitored across a period of rapid spatial learning to assess how the network changes during the initial phases of memory formation and retrieval. In contrast to several reports, the hippocampal network did not display enhanced representation of the goal location via accumulation of place fields or elevated firing rates at the goal. Rather, population activity rates increased globally as a function of experience. These alterations in activity were mirrored in the power of the theta oscillation and in the quality of theta sequences, without preferential encoding of paths to the learned goal location. In contrast, during brief “offline” pauses in movement, representation of a novel goal location emerged rapidly in ripples, preceding other changes in network activity. These data demonstrate that the hippocampal network can facilitate active navigation without enhanced goal representation during periods of active movement, and further indicate that goal representation in hippocampal ripples before movement onset supports subsequent navigation, possibly through activation of downstream cortical networks.SIGNIFICANCE STATEMENT Understanding the mechanisms through which the networks of the brain rapidly assimilate information and use previously learned knowledge are fundamental areas of focus in neuroscience. In particular, the hippocampal circuit is a critical region for rapid formation and use of spatial memory. In this study, several circuit-level features of hippocampal function were quantified while rats performed a spatial navigation task requiring rapid memory formation and use. During periods of active navigation, a general increase in overall network activity is observed during memory acquisition, which plateaus during memory retrieval periods, without specific enhanced representation of the goal location. During pauses in navigation, rapid representation of the distant goal well emerges before either behavioral improvement or changes in online activity.     

Neonatal mice of the down syndrome model, Ts65Dn, exhibit upregulated VIP measures and reduced responsiveness of cortical astrocytes to VIP stimulation

The Ts65Dn segmental mouse model of Down syndrome (DS) possesses a triplication of the section of chromosome 16 that is most homologous to the human chromosome 21 that is trisomic in DS. This model exhibits many of the characteristics of DS including small size, developmental delays, and a decline of cholinergic systems and cognitive function with age. Recent studies have shown that vasoactive intestinal peptide (VIP) systems are upregulated in aged Ts65Dn mice and that VIP dysregulation during embryogenesis is followed by the hypotonia and developmental delays as seen in both DS and in Ts65Dn mice. Additionally, astrocytes from aged Ts65Dn brains do not respond to VIP stimulation to release survival-promoting substances. To determine if VIP dys-regulation is age-related in Ts65Dn mice, the current study examined VIP and VIP receptors (VPAC-1 and VPAC-2) in postnatal day 8 Ts65Dn mice. VIP and VPAC-1 expression was significantly increased in the brains of trisomic mice compared with wild-type mice. VIP-binding sites were also significantly increased in several brain areas of young Ts65Dn mice, especially in the cortex, caudate/putamen, and hippocampus. Further, in vitro treatment of normal neurons with conditioned medium from VIP-stimulated Ts65Dn astrocytes from neonatal mice did not enhance neuronal survival. This study indicates that VIP anomalies are present in neonatal Ts65Dn mice, a defect occurs in the signal transduction mechanism of the VPAC-1 VIP receptor, cortical astrocytes from neonatal brains are dysfunctional, and further, that VIP dysregulation may play a significant role in DS.     

Vasoactive intestinal peptide in the brain of a mouse model for Down syndrome

The most common genetic cause of mental retardation is Down syndrome, trisomy of chromosome 21, which is accompanied by small stature, developmental delays, and mental retardation. In the Ts65Dn segmental trisomy mouse model of Down syndrome, the section of mouse chromosome 16 most homologous to human chromosome 21 is trisomic. This model exhibits aspects of Down syndrome including growth restriction, delay in achieving developmental milestones, and cognitive dysfunction. Recent data link vasoactive intestinal peptide malfunction with developmental delays and cognitive deficits. Blockage of vasoactive intestinal peptide during rodent development results in growth and developmental delays, neuronal dystrophy, and, in adults, cognitive dysfunction. Also, vasoactive intestinal peptide is elevated in the blood of newborn children with autism and Down syndrome. In the current experiments, vasoactive intestinal peptide binding sites were significantly increased in several brain areas of the segmental trisomy mouse, including the olfactory bulb, hippocampus, cortex, caudate/putamen, and cerebellum, compared with wild-type littermates. In situ hybridization for VIP mRNA revealed significantly more dense vasoactive intestinal peptide mRNA in the hippocampus, cortex, raphe nuclei, and vestibular nuclei in the segmental trisomy mouse compared with wild-type littermates. In the segmental trisomy mouse cortex and hippocampus, over three times as many vasoactive intestinal peptide-immunopositive cells were visible than in wild-type mouse cortex. These abnormalities in vasoactive intestinal peptide parameters in the segmental trisomy model of Down syndrome suggest that vasoactive intestinal peptide may have a role in the neuropathology of Down-like cognitive dysfunction.     

Increased Variability and Asymmetric Expansion of the Hippocampal Spatial Representation in a Distal Cue‐Dependent Memory Task

Place cells in the hippocampus fire at specific positions in space, and distal cues in the environment play critical roles in determining the spatial firing patterns of place cells. Many studies have shown that place fields are influenced by distal cues in foraging animals. However, it is largely unknown whether distal‐cue‐dependent changes in place fields appear in different ways in a memory task if distal cues bear direct significance to achieving goals. We investigated this possibility in this study. Rats were trained to choose different spatial positions in a radial arm in association with distal cue configurations formed by visual cue sets attached to movable curtains around the apparatus. The animals were initially trained to associate readily discernible distal cue configurations (0° vs. 80° angular separation between distal cue sets) with different food‐well positions and then later experienced ambiguous cue configurations (14° and 66°) intermixed with the original cue configurations. Rats showed no difficulty in transferring the associated memory formed for the original cue configurations when similar cue configurations were presented. Place field positions remained at the same locations across different cue configurations, whereas stability and coherence of spatial firing patterns were significantly disrupted when ambiguous cue configurations were introduced. Furthermore, the spatial representation was extended backward and skewed more negatively at the population level when processing ambiguous cue configurations, compared with when processing the original cue configurations only. This effect was more salient for large cue‐separation conditions than for small cue‐separation conditions. No significant rate remapping was observed across distal cue configurations. These findings suggest that place cells in the hippocampus dynamically change their detailed firing characteristics in response to a modified cue environment and that some of the firing properties previously reported in a foraging task might carry more functional weight than others when tested in a distal‐cue‐dependent memory task. © 2016 Wiley Periodicals, Inc.     

Reduced phospholipase C-beta activity and isoform expression in the cerebellum of TS65Dn mouse: a model of Down syndrome.

Agonist- and guanine-nucleotide-stimulated phospholipase C-beta (PLC) activity was characterized in crude plasma membrane preparations from cerebral cortex, hippocampus and cerebellum of Ts65Dn mice, a model for Down syndrome, and their control littermates. The levels of expression of PLC-beta((1-4)) isoforms and G-protein alpha(q/11) subunits were also quantified by Western blot analysis to establish their contribution to the patterns of PLC functioning. PLC activity regulated by G-proteins and muscarinic and 5-HT(2) receptors presented a regional distribution in both control and Ts65Dn mice. In both groups of mice, the intensity of PLC responses to maximal activation by calcium followed the sequence cerebellum > cortex > hippocampus. Both basal and maximal PLC activities, however, were significantly lower in cerebellar membranes of Ts65Dn than in control mice. This difference was mostly revealed in crude plasma membranes prepared from cerebellum at the level of G-protein-dependent-PLC activity because the concentration-response curve to GTPgammaS showed a reduction of the maximal effect in Ts65Dn mice, with no change in sensitivity (EC(50)). Western blot analysis showed a heterogeneous distribution of PLC-beta((1-4)) isoforms in both groups of mice. The levels of PLC-beta4 isoform, however, were significantly lower in the cerebellum of Ts65Dn than in control mice. We conclude that the cerebellum of Ts65Dn mice has severe deficiencies in PLC activity stimulated by guanine nucleotides, which are specifically related to a lower level of expression of the PLC-beta4 isoform, a fact that may account for the neurological phenotype observed in this murine model of Down syndrome.     

Abnormal expression of the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) in hippocampus, frontal cortex, and substantia nigra of Ts65Dn mouse: a model of Down syndrome

Ts65Dn, a mouse model of Down syndrome (DS), demonstrates abnormal hippocampal synaptic plasticity and behavioral abnormalities related to spatial learning and memory. The molecular mechanisms leading to these impairments have not been identified. In this study, we focused on the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) gene that is highly expressed in the hippocampus region. We studied the expression pattern of GIRK subunits in Ts65Dn and found that GIRK2 was overexpressed in all analyzed Ts65Dn brain regions. Interestingly, elevated levels of GIRK2 protein in the Ts65Dn hippocampus and frontal cortex correlated with elevated levels of GIRK1 protein. This suggests that heteromeric GIRK1-GIRK2 channels are overexpressed in Ts65Dn hippocampus and frontal cortex, which could impair excitatory input and modulate spike frequency and synaptic kinetics in the affected regions. All GIRK2 splicing isoforms examined were expressed at higher levels in the Ts65Dn in comparison to the diploid hippocampus. The pattern of GIRK2 expression in the Ts65Dn mouse brain revealed by in situ hybridization and immunohistochemistry was similar to that previously reported in the rodent brain. However, in the Ts65Dn mouse a strong immunofluorescent staining of GIRK2 was detected in the lacunosum molecular layer of the CA3 area of the hippocampus. In addition, tyrosine hydroxylase containing dopaminergic neurons that coexpress GIRK2 were more numerous in the substantia nigra compacta and ventral tegmental area in the Ts65Dn compared to diploid controls. In summary, the regional localization and the increased brain levels coupled with known function of the GIRK channel may suggest an important contribution of GIRK2 containing channels to Ts65Dn and thus to DS neurophysiological phenotypes.