Cholinergic dysfunction, neuronal damage and axonal loss in TgCRND8 mice

In 7-month-old TgCRND8 mice, the extracellular cortical acetylcholine levels in vivo, the number and morphology of cholinergic neurons in the nucleus basalis magnocellularis and the ability to acquire an inhibitory avoidance response in the step-down test were studied. The TgCRND8 mouse brain is characterized by many beta-amyloid plaques, reduced neuronal and axonal staining, white matter demyelination, glia reaction and inducible nitric oxide synthase immunoreactivity. Choline acetyltransferase immunoreactivity in the nucleus basalis magnocellularis was significantly decreased. Basal and potassium-stimulated extracellular acetylcholine levels, investigated by microdialysis, and m2 muscarinic receptor immunoreactivity were reduced in the cortex of TgCRND8 mice, and scopolamine administration increased cortical extracellular acetylcholine levels in control but not in TgCRND8 mice. A cognitive impairment was demonstrated in the step-down test. These findings demonstrate that neuronal damage and cholinergic dysfunction in vivo underlie the impairment in learning and memory functions in this mouse model of Alzheimer's disease.     

Age-dependent effects of environmental enrichment on spatial reference memory in male mice

Although environmental enrichment has been shown to improve various types of memory in young and aging mice, no study has directly compared the degree to which enrichment improves memory at different ages throughout the lifespan in male mice. Therefore, the present study investigated the effects of long-term continuous enrichment in young (3 months), middle-aged (15 months), and aged (21 months) male C57BL/6 mice. Spatial reference memory was tested in the Morris water maze. Results demonstrate that 24h/day environmental enrichment for approximately 6 weeks significantly improved spatial memory in the Morris water maze in aged males, but not in young or middle-aged males. These data also indicate that 24h exposure to complex enriched housing conditions increases the magnitude of enrichment-induced improvements in memory among aged mice relative to those previously reported by this lab and others.     

PSEN1 promoter demethylation in hyperhomocysteinemic TgCRND8 mice is the culprit, not the consequence

In recent years, in parallel with the growing awareness of the multifactorial nature of Late Onset Alzheimer's Disease, the possibility that epigenetic mechanisms could be involved in the onset and/or progression of the pathology assumed an increasingly intriguing and leading role in Alzheimer's research. Today, many scientific reports indicate the existence of an epigenetic drift during ageing, in particular in Alzheimer's subjects. At the same time, experimental evidences are provided with the aim to demonstrate the causative or consequential role of epigenetic mechanisms. Our research group was involved in the last ten years in studying DNA methylation, the main epigenetic modification, in relationship to altered one-carbon metabolism (namely high homocysteine and low B vitamins levels), in Alzheimer's experimental models. Our previous findings about the demethylation of Presenilin1 gene promoter in nutritionally-induced hyperhomocysteinemia in a transgenic mouse model clearly demonstrated that Presenilin1 is regulated by DNA methylation. One of the open questions raised by our studies was if the observed demethylation was solely due to the induced imbalance of one-carbon metabolism or could be a response to the massive deposition of amyloid plaques in transgenic mice. Here we analyzed old (10 months) mice under standard diet in order to evidence possible changes in Presenilin1 promoter methylation in transgenic (TgCRND8 mice, carrying a double-mutated human APP transgene) vs. wt mice (129Sv) after prolonged exposure to amyloid. We found no differences in Presenilin1 methylation despite a slight increase in gene expression; these results suggest that amyloid production is not responsible for Presenilin1 demethylation in TgCRND8 mice brain.     

Abnormal processing of tau in the brain of aged TgCRND8 mice

Amyloid plaques and neurofibrillary tangles are the main histopathological hallmarks of Alzheimer's disease (AD). In the neocortex and hippocampus of aged TgCRND8 mice, tau is hyperphosphorylated at different sites recognized by PHF-1, AT100, AT8 and CP13 antibodies. Phospho-SAPK/JNK levels were increased in the tg mouse brain, where activated SAPK/JNK co-localizes with PHF-1-positive cells. Phosphorylated tau-positive cells showed Bielschowsky- and Thioflavine S-positive intraneuronal deposits. PHF-1 and nitrotyrosine immunoreactivity merged within neurons surrounding amyloid deposits in cortical and hippocampal areas and immunoprecipitation studies confirmed that tau is nitrosylated. Our findings, demonstrating the presence of hyperphosphorylated and nitrosylated tau protein as well as of insoluble aggregates after the onset of amyloid deposition in the TgCRND8 mouse brain, indicate that the abnormal processing of tau may occur subsequently to cerebral amyloidosis and that activation of SAPK/JNK and induction of nitrosative stress are the more likely connecting factors between amyloidosis and tauopathy in AD.     

Spatial learning,exploration, anxiety,and motor coordination in female APP23 transgenic mice with the Swedish mutation

Transgenic mice overexpressing the betaAPP gene with the Swedish mutation under the control of the murine thy-1 promoter show Alzheimer-like characteristics including the accumulation of Abeta protein in the cerebral cortex. Female 16-month-old APP23 transgenic mice were compared to age-matched non-transgenic mice in behavioral tests measuring spatial learning, exploration of environmental stimuli, anxiety, and motor coordination. APP23 transgenic mice had fewer fast ambulatory movements, either fast or slow stereotypy movements, and slow rears in a photocell activity chamber. The acquisition of spatial learning in the Morris water maze was impaired in APP23 transgenic mice, but not during the probe test or while swimming towards a visible platform. Neither were there intergroup differences in tests of anxiety or motor coordination. These results indicate that a learning deficit and hypoactivity, concordant with the early stages of Alzheimer's disease, characterize this mouse model with Abeta accumulation.     

Effects of continuous and intermittent estrogen treatments on memory in aging female mice

The manner in which hormone therapy is given to postmenopausal women may significantly influence its ability to reduce age-associated memory loss. To test the hypothesis that a regimen that approximates the timing of estrogen surges in the natural cycle is more beneficial for memory than a regimen that provides continuous levels of estrogen, we examined the effects of continuous and intermittent estrogen regimens on spatial and object memory in aging female mice. Mice (18 months) were treated with 0.2 mg/kg 17beta-estradiol (E(2)) or vehicle (VEH) for 3 months following ovariectomy. A fast-acting water-soluble cyclodextrin-encapsulated E(2) was used to ensure metabolism within 24 h. Vehicle-treated mice received daily injections of 2-hydroxypropyl-beta-cyclodextrin vehicle. The continuous estradiol group (Contin E(2)) was injected daily with estradiol. The intermittent group (Twice/wk E(2)) received estradiol every 4 days and vehicle on all other days. Mice (21 months) were tested in water-escape motivated 8-arm radial arm maze (WRAM) and object recognition tasks. During WRAM acquisition, the Twice/wk E(2) group committed significantly more reference memory errors than VEH and Contin E(2) groups, and tended to make more working memory errors than the VEH group. The Contin E(2) group did not differ from VEH on either WRAM measure. Additionally, the Twice/wk E(2) group tended to exhibit impaired object recognition. Thus, neither treatment improved spatial or object memory. Indeed, intermittent estradiol was detrimental to both types of memory. These results suggest that the timing of administration may play an important role in the mnemonic response of aging females to estrogen.     

Long-lasting and transgenerational effects of an environmental enrichment on memory formation

It has long been believed that genetically determined, but not environmentally acquired, phenotypes can be inherited. However, a large number of recent studies have reported that phenotypes acquired from an animal's environment can be transmitted to the next generation. Moreover, epidemiology studies have hinted that a similar phenomenon occurs in humans. This type of inheritance does not involve gene mutations that change DNA sequence. Instead, it is thought that epigenetic changes in chromatin, such as DNA methylation and histone modification, occur. In this review, we will focus on one exciting new example of this phenomenon, transfer across generations of enhanced synaptic plasticity and memory formation induced by exposure to an "enriched" environment.     

Heterozygous mice with Ric-8 mutation exhibit impaired spatial memory and decreased anxiety

Ric-8 is a guanine nucleotide exchange factor for a subset of Galpha proteins and it is required to maintain Galpha(q) and the Galpha(s) pathways in functional state. In adult mice Ric-8 is expressed in regions involved in the regulation of behavior (neocortex, cingulate cortex and hippocampus). As Ric-8 is shown to regulate neuronal transmitter release, the aim of present study was to perform behavioral analysis of ric-8 mutant. Homozygous (-/-) ric-8 mutant mice are not viable and die in early embryonic development, therefore for behavioral analysis heterozygous (+/-) ric-8 mutant mice were used. We found decreased anxiety of ric-8 heterozygous mice in light-dark compartment test where mutant mice significantly avoided the light compartment. In spatial learning paradigm (Morris water maze) the performance of ric-8 (+/-) mice was impaired. Namely, in the reversal test, ric-8 (+/-) mice exhibited significant delay to find the hidden platform compared to wild-type (wt) littermates. We did not find differences in the behavioral tests reflecting the motor abilities of mice (motor activity, rota-rod). Therefore, described alterations seem to be specific for anxiety and spatial learning. Based on these results we can conclude the importance of ric-8 in the regulation of memory and emotional behavior.     

Effects of physostigmine and lecithin on memory in Alzheimer disease

Because there is evidence that central cholinergic mechanisms are depleted in dementia, we studied the effects of central cholinergic augmentation on the memory of 5 patients with Alzheimer disease. Patients received placebo, lecithin, physostigmine, or lecithin plus physostigmine in a double-blind study using titrated doses of the acetylcholinesterase inhibitor physostigmine. Memory was evaluated with alternate forms of the selective reminding procedure. Compared with lecithin alone, the combination of physostigmine and lecithin consistently enhanced memory storage and retrieval; physostigmine without lecithin produced no memory facilitation. The strategy of combining a cholinergic agonist and precursor holds promise, although a larger clinical trial is needed.     

Cage-induced stereotypies, perseveration and the effects of environmental enrichment in laboratory mice

When kept in barren and restrictive cages, animals frequently develop stereotypic behaviour patterns that are characterized by high repetition rates, conspicuous invariance and an apparent lack of function. Although millions of animals are affected, the underlying causes and mechanisms are still unclear. Growing evidence suggests that cage-induced stereotypies may reflect pathological dysfunction within basal ganglia circuitry expressed by perseverative behaviour. In order to assess whether variation in stereotypy performance and variation in perseverative behaviour may have a common cause in ICR CD-1 mice, we assessed the effects of environmental enrichment on both phenomena. We raised 48 female ICR CD-1 mice in standard or enriched cages from three weeks to either 6 or 11 months of age and measured stereotypy level in the home cage and perseveration on an extinction task. We further examined whether enriched rearing conditions (early enrichment) protect mice from the developing stereotypies later in life and whether stereotypies developed in barren cages would persist in an enriched environment (late enrichment) by transferring standard mice to enriched cages and vice versa for 14 weeks after completion of the extinction task. We found no evidence for a causal relation between stereotypy and perseveration in mice. However, transfer to enriched cages reduced stereotypy levels significantly both at 6 and 11 months of age indicating that stereotypies had not become established yet. Finally, we found that removing enrichments at both ages did not induce higher stereotypy levels, thereby confirming earlier reports of a neuroprotective effect of early enrichment.     

Effect of environmental enrichment on the immunoendocrine aging of male and female triple-transgenic 3xTg-AD mice for Alzheimer's disease

We have previously shown that 3xTgAD mice (triple-transgenic mice for Alzheimer's disease, harboring PS1(M146V), AβPP(Swe), tau(P301L) transgenes) suffer detrimental changes in some key lymphocyte functions, described as health and longevity markers, with males being more affected than females and showing higher mortality rates. In the present work, 3xTgAD and wild type 129/C57BL6 male and female non- and environmentally enriched mice were used. The enriched environment (EE) began in the adulthood (6 months) and lasted for 5.5 months. The animals were sacrificed at advanced stages of the disease (15 month-old), and spleen, thymus, and plasma were obtained. The results indicate that 3xTg-AD males are especially benefitted from EE exposure, as shown by the improvement in lymphocyte functional activities such as chemotaxis and natural killer cytotoxicity, as well as in plasma corticosterone levels. By contrast, wild type females seem to be highly sensitive to EE removal, as regards the proliferation capacity of lymphocytes and their intracellular glutathione content. These results support the relevance of gender differences in AD when screening for new strategies for the control of the disease, and suggest that active life, by means of EE, should be maintained until natural death in order to preserve all the positive effects that this strategy exerts on the immune system.     

Differential effects of environmental enrichment on behavior and learning of male and female Ts65Dn mice,a model for Down syndrome

We have assessed the effects of enriched environment (EE) upon behavioral and cognitive performances of partially trisomic Ts65Dn (TS) mice and their control (CO) littermates. Enriched environment was applied to pups for 7 weeks after weaning. Circadian spontaneous activity (actimetry), exploratory behavior (hole board), activity in the open field and spatial memory (Morris Water Maze, repeated acquisition and cued paradigms) were analyzed in 86 female and 75 male mice, starting 15 days after completing enrichment. For each gender, mice were distributed in non-enriched and enriched control and trisomic groups. Enriched environment reduced in trisomic females and enhanced in trisomic males' circadian activity. Exploratory behavior was increased by enrichment in all groups, regardless of gender or presence of trisomy. In the Morris Water Maze, a significant improvement of the spatial memory was observed in enriched-control females, but not in enriched-control male mice, as assessed by distances traveled. Performances in the four groups of control animals were also consistently and significantly better than those of matching trisomic mice. In the acquisition trials, enrichment improved performance in trisomic female animals, but deteriorated in trisomic male mice. In all groups, changes in escape latencies and distances induced by enrichment were accounted for by changes in the total time spent in the periphery of the pool, indicating changes in learning strategy. Working memory was the function more affected by enrichment. It is concluded that enriched environment induces behavioral and learning changes in trisomic mice, although gender plays a significant modulatory role.     

Effects of age and anxiety on learning and memory

This study aims to investigate the effects of age and anxiety on behavior, learning and memory in rats. Before and after the anxiety and learning tests, locomotor activity, exploratory activity and autonomic functions of the rats were tested in open field area. At the beginning and at the end of behavior tests, urines were collected so as to determine 5-hydroxyindolaceticacid (5-HIAA) levels. Following these tests, rats were anesthetized and their serum corticosteron (CORT) levels were analyzed. After anxiety, except for defecation, all parameters in open field such as line crossing, rearing, sitting and number of grooming were decreased in both young and aged animals. 5-Hydroxyindolaceticacid levels were decreased and serum CORT levels were increased, it is supported that especially the aged rats were much more affected from anxiety compared to the young ones. Elevated T-maze results show that emotional learning did not change while conditioned performance was tested in the closed arm and unconditioned performance was tested in the open arm. Nevertheless, it is observed that aging leaded to extensions in avoidance responses and thus caused difficulty in learning. In water maze test, rats showed higher performance in reaching the platform in repetitive trials; this demonstrates that they have learned by environmental cues. Experimental group had not better performance in reaching the platform according to control group, so this supports that anxiety affects spatial learning. As a conclusion, it could be stated that especially in aged rats, anxiety that is created by elevated T-maze and cat odor and supported with 5-hydroxyindoleacetic acid and serum corticosterone, causes difficulty in emotional and spatial learning.     

Rosiglitazone attenuates learning and memory deficits in Tg2576 Alzheimer mice

The thiazolidinediones, such as rosiglitazone, increase peripheral insulin sensitivity and their use is proposed for the treatment of Alzheimer's disease. However, the mechanisms underlying the potential beneficial effects of rosiglitazone in Alzheimer's disease remain unclear. In previous studies, we observed that Tg2576 Alzheimer mice develop peripheral insulin resistance with age and have much higher serum corticosterone levels than wild-type mice when fasted overnight. We further showed that both of these defects can be ameliorated by rosiglitazone administration. Here, we report that during behavioral testing which involves repetitive overnight fasting, Tg2576 mice administered rosiglitazone exhibited better spatial learning and memory abilities and had lower serum corticosterone levels than untreated Tg2576 mice. When untreated Tg2576 mice were administered metyrapone, a drug that blocks glucocorticoid production, their spatial learning and memory abilities and serum corticosterone levels were similar to those of rosiglitazone-treated mice. We further report here that rosiglitazone attenuated reductions in insulin-degrading enzyme (IDE) mRNA and activity, and reduced amyloid beta-peptide (Abeta)42 levels without affecting amyloid deposition, in the brains of Tg2576 mice. These results demonstrate that rosiglitazone attenuates learning and memory deficits in Tg2576 mice and suggest that the effects of the drug on learning and memory, brain IDE levels, and brain Abeta42 levels in the mice may be due to its glucocorticoid-lowering actions.     

Environmental enrichment in mice decreases anxiety, attenuates stress responses and enhances natural killer cell activity

The importance of environment in the regulation of brain, behaviour and physiology has long been recognized in biological, social and medical sciences. Animals maintained under enriched conditions have clearly been shown to have better learning abilities than those maintained under standard conditions. However, the effects of environmental enrichment (EE) on immunity and emotionality have been less documented and remain questionable. Therefore, we investigated the effect of EE on natural killer (NK) cell activity, psychological stress responses and behavioural parameters. Male C3H mice were housed either in enriched or standard conditions for 6 weeks. Behaviour was then examined by the grip-strength test, staircase and elevated plus maze, and corticosterone levels and NK cell activity were measured. Furthermore, animals exposed to the stress paradigm, achieved by electric shock with reminders, were tested for freezing time in each reminder. Corticosterone levels were also measured. The EE mice showed decreased anxiety-like behaviour and higher activity compared to standard mice, as revealed by a greater percentage of time spent in the open arms of the elevated plus maze, and a higher rate of climbing the staircase. A shorter freezing time in the stress paradigm and no corticosterone level reactivity were measured in EE mice. In addition, NK cell activity in spleens of EE mice was higher than that demonstrated in those of standard mice. Thus, EE has a beneficial effect on anxiety-like behaviour, stress response and NK cell activity. The effect on NK cell activity is promising, due to the role of NK cells in host resistance.