Carmen Martínez-Cué

Impaired short- and long-term memory in Ts65Dn mice, a model for Down syndrome

Ts65Dn (TS), control littermates (CO) and Swiss (SW) male mice were tested in the elevated plus-maze and in the Morris water maze (MWM) for memory evaluation. In the plus-maze, each mouse was placed at the end of an open arm and initial freezing and the time to enter into an enclosed arm (transfer latency) were measured. SW mice decreased both measures over repeated trials, whereas no decrease of freezing was observed in CO mice, thus suggesting increased emotionality in this group. Compared to CO mice, TS mice showed less initial freezing, shorter transfer latencies, and spent less time in enclosed arms, suggesting a reduced ability to habituate or to inhibit behaviour. Animals were also submitted to a learning-set paradigm consisting of reaching a new platform position each day in the MWM. Two training phases (separated by a resting period of 6 weeks), each including eight acquisition and four cued sessions, were performed (each session consisting of four pairs of trials). CO and SW mice already reached an asymptotic performance by the second day of the first phase whereas TS mice did not achieve that level until the second training phase. The progression over trials indicated that CO and SW animals learned the new platform position between trials 1 and 2 of each session, whereas TS animals failed to do it and had more difficulties to find the platform when it was placed in the centre of the pool as compared to the other positions (SW, NE, E). The results suggest that TS mice show working memory impairments in addition to long-term memory deficits, although extensive training appeared to facilitate TS mice to achieve a level of performance similar to their control littermates. This represents another aspect of the cognitive deficits shown by TS mice: a mouse model of the human Down syndrome.

Transforming Growth Factors β Coordinate Cartilage and Tendon Differentiation in the Developing Limb Mesenchyme

Transforming growth factor β (TGFβ) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFβ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFβs shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFβ signaling up-regulates Sox9 in the in vivo experimental model system in which TGFβ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFβ signaling, we found that TGFβs appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFβ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFβ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFβ treatments.

Chronic pentylenetetrazole but not donepezil treatment rescues spatial cognition in Ts65Dn mice, a model for Down syndrome

The most commonly used model of Down syndrome, the Ts65Dn (TS) mouse, is trisomic for most of the region of MMU16 that is homologous to HSA21. This mouse shares many phenotypic characteristics with people with Down syndrome including behavioral and cognitive alterations. The objective of this study was to analyze the ability of two drugs that improve cognition in different experimental models, the acetylcholinesterase inhibitor donepezil and the non-competitive GABA(A) antagonist pentylenetetrazole (PTZ), to improve the cognitive deficits found in TS mice. The drugs were administered p.o. to TS and CO mice for 8 weeks and a behavioral characterization was performed. Sensorimotor abilities, including vision, hearing, strength and motor coordination, as well as locomotor activity in the home cage, were not modified by any chronic treatment in TS and CO mice. TS mice showed altered equilibrium in the aluminium rod, and this effect was larger under PTZ treatment. This result may indicate a potential adverse effect of PTZ in Ts65Dn mice. Learning and memory were evaluated in TS and CO mice after both treatments in the Morris water maze. Donepezil administration did not modify learning and memory in animals of any genotype. On the other hand, PTZ administration rescued TS performance in the Morris water maze.

Reducing GABAA α5 Receptor-Mediated Inhibition Rescues Functional and Neuromorphological Deficits in a Mouse Model of Down Syndrome

Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mouse model of DS. We show that chronic treatment of these mice with RO4938581 (3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABAA α5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment suppressed the hyperactivity observed in TS mice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and support the potential therapeutic use of selective GABAA α5 NAMs to treat cognitive dysfunction in DS.

Mouse Models of Down Syndrome as a Tool to Unravel the Causes of Mental Disabilities

Down syndrome (DS) is the most common genetic cause of mental disability. Based on the homology of Hsa21 and the murine chromosomes Mmu16, Mmu17 and Mmu10, several mouse models of DS have been developed. The most commonly used model, the Ts65Dn mouse, has been widely used to investigate the neural mechanisms underlying the mental disabilities seen in DS individuals. A wide array of neuromorphological alterations appears to compromise cognitive performance in trisomic mice. Enhanced inhibition due to alterations in GABAA-mediated transmission and disturbances in the glutamatergic, noradrenergic and cholinergic systems, among others, has also been demonstrated. DS cognitive dysfunction caused by neurodevelopmental alterations is worsened in later life stages by neurodegenerative processes. A number of pharmacological therapies have been shown to partially restore morphological anomalies concomitantly with cognition in these mice. In conclusion, the use of mouse models is enormously effective in the study of the neurobiological substrates of mental disabilities in DS and in the testing of therapies that rescue these alterations. These studies provide the basis for developing clinical trials in DS individuals and sustain the hope that some of these drugs will be useful in rescuing mental disabilities in DS individuals.

Memantine Normalizes Several Phenotypic Features in the Ts65Dn Mouse Model of Down Syndrome

Ts65Dn (TS) mice exhibit several phenotypic characteristics of human Down syndrome, including an increased brain expression of amyloid-beta protein precursor (AbetaPP) and cognitive disturbances. Aberrant N-methyl-D-aspartate (NMDA) receptor signaling has been suspected in TS mice, due to an impaired generation of hippocampal long-term potentiation (LTP). Memantine, an uncompetitive NMDA receptor antagonist approved for the treatment of moderate to severe Alzheimers disease, is known to normalize LTP and improve cognition in transgenic mice with high brain levels of AbetaPP and amyloid-beta protein. It has recently been demonstrated that acute injections of memantine rescue performance deficits of TS mice on a fear conditioning test. Here we show that oral treatment of aged TS mice with a clinically relevant dose of memantine (30 mg/kg/day for 9 weeks) improved spatial learning in the water maze task and slightly reduced brain AbetaPP levels. We also found that TS mice exhibited a significantly reduced granule cell count and vesicular glutamate transporter-1 (VGLUT1) labeling compared to disomic control mice. After memantine treatment, the levels of hippocampal VGLUT1 were significantly increased, reaching the levels observed in vehicle treated-control animals. Memantine did not significantly affect granule cell density. These data indicate that memantine may normalize several phenotypic abnormalities in TS mice, many of which--such as impaired cognition--are also associated with Down syndrome and Alzheimers disease.

Behavioral, cognitive and biochemical responses to different environmental conditions in male Ts65Dn mice, a model of Down syndrome

Ts65Dn mouse is the most widely accepted model for Down syndrome. We previously showed that environmental enrichment improved spatial learning in female but deteriorated it in male Ts65Dn mice. This study analyzed the factors contributing to the disturbed cognition of male Ts65Dn mice after enriched housing, by allocating male control and Ts65Dn mice in four conditions after weaning: small (n = 2-3) and large group (n = 8-10) housing, and enriched housing in small (2-3) and large groups (8-10). Learning, aggressive behavior, anxiety-like behavior and biochemical correlates of stress were evaluated when Ts65Dn and control mice were 4-5 months old. Environmental enrichment in large mixed colonies of Ts65Dn and diploid littermates disturbed behavioral and learning skills of Ts65Dn mice in the Morris water maze. ACTH and testosterone levels were not modified in any group of mice. Ts65Dn and control mice subjected to enriched housing in large groups and Ts65Dn mice housed in large groups showed higher corticosterone levels. Aggressive behavior was evaluated by measuring the number of attacks performed in the presence of an intruder. Ts65Dn mice performed less attacks than controls in all conditions, especially after enriched housing, indicating subordination. In the plus maze, cognitive aspects (i.e. risk assessment) and motor components (open arm avoidance) of anxiety behavior were evaluated; no difference in any condition was found. It is suggested that an excess of social and/or physical stimulation in Ts65Dn mice may affect cognition by disturbing the emotional and behavioral components of the learning process.

Cell proliferation is reduced in the dentate gyrus of aged but not young Ts65Dn mice, a model of Down syndrome.

Reduced number of neurons is a common feature in Downs syndrome (DS) brains. Since reduced neuronal number also occurs in the dentate gyrus of Ts65Dn mice (TS), a model for DS, hippocampal cell proliferation and survival were analyzed in young and old TS mice. For evaluating proliferation and survival, half of the mice were sacrificed 1 day, and the other half 30 days after the last bromodeoxyuridine injection, respectively. No difference was found in the number of proliferating or surviving cells of young TS and control mice. An age-associated decline in total cell number and density has been found in both genotypes, this decline being more pronounced in TS animals. Thus, aged TS mice showed reduced cell proliferation and density of surviving cells compared to CO mice. Due to the putative involvement of newborn cells in the dentate gyrus in learning processes, the reduced proliferative capacity found in TS mice could be involved in the cognitive problems found in this model of Down syndrome.

Both increases in immature dentate neuron number and decreases of immobility time in the forced swim test occurred in parallel after environmental enrichment of mice.

A direct relation between the rate of adult hippocampal neurogenesis in mice and the immobility time in a forced swim test after living in an enriched environment has been suggested previously. In the present work, young adult mice living in an enriched environment for 2 months developed considerably more immature differentiating neurons (doublecortin-positive, DCX(+)) than control, non-enriched animals. Furthermore, we found that the more DCX(+) cells they possessed, the lower the immobility time they scored in the forced swim test. This DCX(+) subpopulation is composed of mostly differentiating dentate neurons independently of the birthdates of every individual cell. However, variations found in this subpopulation were not the result of a general effect on the survival of any newborn neuron in the granule cell layer, as 5-bromo-2-deoxyuridine (BrdU)-labeled cells born during a narrow time window included in the longer lifetime period of DCX(+) cells, were not significantly modified after enrichment. In contrast, the survival of the mature population of neurons in the granule cell layer of the dentate gyrus in enriched animals increased, although this did not influence their performance in the Porsolt test, nor did it influence the dentate gyrus volume or granule neuronal nuclei size. These results indicate that the population of immature, differentiating neurons in the adult hippocampus is one factor directly related to the protective effect of an enriched environment against a highly stressful event.

Impaired cyclic AMP production in the hippocampus of a Down syndrome murine model

Behavioral and learning disturbances have been found in mice with partial trisomy 16, a new model for Down syndrome. Basal production of cyclic AMP in the hippocampus of trisomic mice was shown to be impaired. In addition, the responses of adenylyl cyclase to the stimulation of beta-adrenoceptors with isoprenaline and of the catalytic subunit with forskolin were both severely depressed.