Enriched Environment (EE) effects on cognitive aging: system memory consolidation in aged EE mice and effects of physical and cognitive training in human subject with Mild Cognitive Impairment (MCI)

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(1)UNIVERSITY OF PISA. PhD Course in Basic and Developmental Neuroscience SSD M-PSI/02. Enriched Environment (EE) effects on cognitive aging: system memory consolidation in aged EE mice and effects of physical and cognitive training in human subject with Mild Cognitive Impairment (MCI). Tutors. Candidate. Prof.ssa Nicoletta Berardi. Dott.ssa Simona Cintoli. Dott. Matteo Caleo. CYCLE 2011-2013.

(2) INDEX FOREWORD ..................................................................................................................................3 INTRODUCTION .........................................................................................................................5 COGNITIVE AGING .................................................................................................................5 COGNITIVE AND PHYSICAL STIMULATION: STUDIES IN HUMANS .....................13 ENRICHED ENVIRONMENT: ANIMAL STUDIES...........................................................17 MEMORY FORMATION AND CONSOLIDATION ..........................................................20 AIM OF THE THESIS ..............................................................................................................27 MATERIALS AND METHODS ...............................................................................................31 TRAIN THE BRAIN .................................................................................................................31 Train the Brain - Study protocol ............................................................................................31 The screening evaluation ........................................................................................................34 Neuropsychological assessment..............................................................................................36 Statistics .................................................................................................................................37 ANIMAL TREATMENT .........................................................................................................39 Assessment of EE effects on system consolidation in young mice .........................................39 Assessment of EE effects on system consolidation in aged mice ............................................39 BEHAVIOURAL TESTS ..........................................................................................................41 Morris Water Maze (MWM) .................................................................................................41 Socially transmitted food preference (STFP) .........................................................................41 IMMUNOHISTOCHEMISTRY ..............................................................................................45 Analysis of c-Fos Positive Cells..............................................................................................45 Analysis of immunohistochemical signal of histone H3 acetylation in the PFC ...................46 STATISTICS ..............................................................................................................................48 RESULTS ......................................................................................................................................49 TRAIN THE BRAIN .................................................................................................................49 Cognitive and physical training improves global cognitive status ........................................52 Cognitive and physical training reduces the neuropsychiatric disturbances ........................61 Cognitive and physical training have no effects on an activities of daily living ....................64 SYSTEM CONSOLIDATION OF SPATIAL MEMORIES IN MICE: EFFECTS OF ENRICHED ENVIRONMENT ...............................................................................................66 Hippocampus is activated following recent and remote spatial memory recall .....................66 EE induces an early recruitment of the mPFC ......................................................................70 EE induces the involvement of a distributed cortical network supporting remote spatial memory ...................................................................................................................................72 SYSTEM CONSOLIDATION IN AGED MICE: EFFECTS OF ENRICHED ENVIRONMENT .....................................................................................................................75 Hippocampus is activated following recent and remote memory recall .................................75 The Orbitofrontal cortex is activated following remote memory in EE mice .........................79 DISCUSSION ..............................................................................................................................84 REFERENCES ..............................................................................................................................93. . 2 .

(3) FOREWORD Brain aging is a complex physiological process which includes an age related cognitive decline, particularly evident for long term declarative memory, working memory, attention and processing speed. There is a large literature of epidemiological studies in humans which suggests that lifestyle factors, such as physical activity, cognitive activity and social interactions, factors which are the main components of an “enriched environment”, can be protective against the development of pathological cognitive decline in elders and reduce the incidence of dementia. In parallel, studies in animals have shown that exposure to an Enriched Environment markedly improves memory performance in aged animals. This would suggest that interventions based on the “enriched environment” paradigm might benefit the cognitive status of elders. However, the literature reports no study investigating the effects of an intervention based on the combination of cognitive and physical training in aged humans already showing signs of cognitive impairment. In Pisa such a study is currently active and is the project “Train the Brain”, employing a combination of physical exercise, cognitive training and stimulation in a social setting to test whether such a non-pharmacological treatment is effective in decreasing the rate of cognitive impairment or even in obtaining a cognitive enhancement in subject with Mild Cognitive Impairment (MCI), considered subjects at risk for developing dementia. During my PhD, within the project “Train the Brain”, I have analysed the effects of 7 months of a physical and cognitive training program in a social setting in subject with MCI using a neuropsychological battery to assess the effects of the intervention at the end of the 7 months and in subsequent follow-up tests. In parallel I have addressed the possible mechanisms of action of an Enriched Environment on cognitive aging exploiting animal models. The formation of long term declarative memories is a process mediated by the hippocampus and other medial temporal lobe structures and relies upon plasticity mechanisms in these structures, which allows a first, local, consolidation of the memory trace; however, it has been recently shown that for context-rich declarative memories, such as episodic memory, the successful recall of memory traces at some temporal . 3 .

(4) distance from their formation requires the additional contribution of neocortical structures, which are progressively recruited for memory storage and recall in a process called memory system consolidation. A crucial mechanism underlying memory system consolidation consists in the functional and structural reorganisation of the pattern of cortical region activation prompted by the reactivation of hippocampal-cortical pathways and the strengthening of cortico-cortical connections, and with the fundamental involvement of cortical plasticity processes. While age related changes in hippocampal plasticity which might underlie age related changes in long-term declarative memory formation have been deeply investigated, no attempt has yet been done to investigate whether system consolidation is impaired by brain aging. Surprisingly, system consolidation has not even been considered as a possible target of the action of enriched environmental conditions in enhancing brain plasticity and improving memory performance in adult or aged animals. I have first tested whether Enriched Environment (EE) could affect the system consolidation process in adult mice: in particular, I investigated whether EE could accelerate the time course of the activation of neocortical areas, possibly through a strengthening of the hippocampal-cortical and the cortico-cortical connections. To do so, I characterized the time course of hippocampal and cortical activation following spatial learning in a brain-wide manner following recalls delayed up to 50 days, using the expression of c-Fos protein as an indicator of neuronal activity in the different brain regions examined. In a second experiment I studied whether EE could affect memory system consolidation in aged mice. To pinpoint the post-learning mechanisms underlying the hippocampal-cortical dialogue during the course of system-level memory consolidation, I trained aged mice, either left in standard cages or put for 40 days in EE, in the social transmission of food preference test (STFP), which involves an ethologically based form of associative olfactory memory. Mice have been tested for memory retrieval either 1 day (recent memory) or 30 days (remote memory) later and system consolidation investigated assessing the pattern of brain area activation and the state of histone acetylation in prefrontal cortical areas. . . . 4 .

(5) INTRODUCTION COGNITIVE AGING Aging is a physiological condition of life, accompanied by several changes in sensory, motor and cognitive functions. There is considerable variance in the degree of cognitive decline associated with non pathological aging, with some individuals maintaining strikingly good intellectual abilities well into, and even beyond, their nineties. While some of the factors associated with graceful aging seem to be linked to genetic endowment, many others are linked to environmental factors, such as the subject lifestyle, and could be modifiable even in middle age to promote a successful aging (Sale et al., 2014). Age-related cognitive performance declines are particularly evident in information processing speed, in orientation tasks, in working memory tasks and in declarative memory tasks, tasks involving the prefrontal cortex and the hippocampus and other medial temporal lobe structures. Also performance in inhibitory functions and in attention tasks, relying on prefrontal cortex, declines with age (Park and Reuter-Lorenz, 2009). Similarly, animal models of aging show a decline in medial temporal lobe dependent and prefrontal cortex dependent memory (Burke and Barnes, 2006). Many studies point out that agerelated changes in cognition cannot be accounted for by a generalized loss of neurons but rather by specific functional and morphological alterations occurring in the medial temporal lobe and the prefrontal cortex, such as agerelated reduction in synaptic density and neuropil, resulting in reduction in volume of specific brain structures, with some regions showing also a loss of neurons (Burke and Barnes, 2006; Berchtold et al., 2008; Holden and Gilbert, 2012). In addition, there is an alteration in synaptic function and plasticity with age, with long term hippocampal synaptic plasticity, crucial for the formation and local consolidation of declarative memories, showing a conspicuous, age dependent, impairment (Burke and Barnes, 2006), and a reduction of hippocampal neurogenesis (Kempermann et al., 1997; 1998; 2002; Spalding et al., 2013).. . 5 .

(6) So, normal aging causes evident changes at multiple levels; in particular, aging alters forms of synaptic plasticity related to declarative memory processes and strongly reduces hippocampal neurogenesis. In parallel to examples of diminished neural plasticity with age, there are numerous examples of what can be considered compensatory plasticity during the aging process. This shows up mostly in terms of a larger and more elaborate pattern of activation of brain areas, and in particular of the prefrontal cortex, in aged with respect to young subjects performing the same task (Park and ReuterLorenz, 2009): the idea is that the aging brain can maintain a relatively high level of performance through neural plasticity processes, optimizing goal attainment with the available resources. Very different is of course the situation in presence of age-associated neurodegenerative diseases leading to dementia, such as Alzheimer’s disease (AD). AD affects an increasing proportion of individuals, imposing a huge cost to our society. Most common causes of dementia are Alzheimer's Disease (AD) and vascular dementia (VD). No treatment is available: drugs currently used are poorly effective and do not prevent, heal, stop, or delay the progression of the disease. It is therefore crucial to find interventions and lifestyle factors to counteract and slow down cognitive decline from an early stage. The pathophysiological process of AD is known to begin many years, if not decades, before the clinical diagnosis of dementia (Morris, 2005). This long “preclinical” phase of AD would provide a critical opportunity for therapeutic intervention (Fig. 1). It is now hypothesised the existence of a long transition phase between normal aging and dementia with moderate levels of cognitive impairment and the presence of subtle cerebral alterations preceding of several years the clinical onset of the disease (Jones et al., 2004; DeCarli et al., 2007; Pike et al., 2007; Garibotto et al., 2008). This early stage of the disease is referred to as Mild Cognitive Impairment (MCI): a clinical diagnosis in which deficits in cognitive function are evident but not of sufficient severity to warrant a diagnosis of. . 6 .

(7) dementia. Physiological cerebral aging and MCI represent different stages along a continuum that may eventually evolve into overt dementia. This prodromal phase is detectable with techniques such as structural and functional neuroimaging, cerebrospinal fluid (CSF) assays, and other biomarkers able to detect evidence of the AD pathophysiological process in vivo showed that biomarker evidence of amyloid beta (Aβ) accumulation, which is associated with functional and structural brain alterations can be observed in clinically normal older individuals with patterns of abnormality like those seen in patients with MCI and AD dementia (Fig. 2). In particular, MCI is characterized by objective deficits in one single domain or multiple domains of cognition, which do not yet configure as overt dementia. A wide variety of neuropsychological tests are extremely useful in making the MCI diagnosis and tracking the evolution of cognitive symptoms over time (Cullen et al., 2007; Nelson and O’Connor, 2008; Petersen et al., 1999; 2008; Tab.1). The rate of yearly progression to dementia of MCI subjects is much higher than in the non-MCI elderly: in particular, the amnestic subtype of MCI (aMCI) may represent a prodromal form of AD. However, a good proportion of MCI subjects not only do not develop dementia, but can also recover from the initial slight impairments that characterizes them as MCI subjects (Jack et al., 2005; Frisoni et al., 2004; Small et al., 2007). Another condition, which has drawn much interest, is the Subjective cognitive impairment (SCI), a very common complaint in the older community population. SCI can be reported in the younger population as episodes of transient confusion, while in the older people is mainly referred as forgetfulness or needing to take notes (Ginó et al., 2010). Some Authors observed a five-fold greater likelihood of decline to MCI or dementia, over a 7-year mean follow-up interval, in people with subjective complaints compared with similarly aged individuals who are free of subjective complaints of impairment (Reisberg et al., 2005). Although a number of studies have reported associations between SCI and underlying brain changes including smaller hippocampal volume (van Norden et al., 2008; Stewart et al., 2008) functional magnetic resonance imaging. . 7 .

(8) correlates (Rodda et al., 2011; Haley, 2011) and evidence of Aβ on brain imaging (Perrotin et al., 2012; Merrill et al., 2012), SCI’s positive predictive value in clinical practice remains low (Palmer et al., 2003). From these considerations, it is clear that the detection of predictive factors of cognitive decline would be very useful in clinical practice. As. previously. said,. cognitive. decline. with. age. shows. a. great. interindividual variability, and, recently, many epidemiological studies have highlighted, as protective factors for developing dementia with age, lifestyle factors, such as education, work type, being engaged in cognitively stimulating and social activities and practicing physical exercise (Laurin et al., 2001; Fratiglioni et al. 2004; Kramer and Erickson, 2007). The first protective factor examined in the literature, and which included education and work type, has been the Cognitive Reserve (CR). The concept of cognitive reserve postulates that subjects with a greater cognitive reserve can sustain a larger amount of brain damage before reaching the threshold for a clinical expression of cognitive impairment. Cognitive reserve is an active reserve and encompasses increased cognitive function and enhanced complex mental activity as protective factors against major age-related cognitive decline and dementia (Stern, 2009). Amongst the factors possibly contributing to cognitive reserve development, education, occupational attainments and leisure activity have been shown, by epidemiological studies, to provide reserve capacity against the effects of aging and disease on brain function (Katzman, 1993; Riley et al., 2005; Valenzuela and Sachdev, 2006). Separate or synergistic effects for higher educational and occupational attainment and leisure activities have been demonstrated, suggesting that each of these life experiences contributes independently to the reserve (Stern, 2009). Several epidemiological studies have provided support for the cognitive reserve hypothesis and in particular for the protective effects of education; as an example, Katzman has shown that subjects with high levels of education have a 5 years delay in dementia onset (Katzman, 1993). The Nun study (Riley et al., 2005) has shown that the presence of elevated levels of ideation and creativity, as estimated from youth autobiographies written at entrance in the convent,. . 8 .

(9) correlated with maintenance of good cognitive capacities in old age: nuns well in their 80’s who had an intact memory were those with the highest ideation capacity. Yaffe and colleagues followed the time-course of cognitive status of 2509 well-functioning elders enrolled in a prospective study for 7 years: characteristics of the cognitive reserve, such as education level, literacy and life style, resulted significant predictors of being a maintainer versus a minor decliner, according to the cognitive score change with time (Yaffe et al., 2009). Other Authors have demonstrated that CR, as well as cognitively stimulating activities, may prevent or slow deposition of Aβ (Landau et al., 2012) and is able to influence the onset and progression of clinical symptom of AD (Wilson et al., 2013). Additional evidence has been provided by neuroimaging methods: functional imaging in vivo such as PET has shown a greater degree of impairment in regional cerebral metabolic activity and blood flow, for a given level of dementia, in highly educated patients (Alexander et al., 1997). Amyloid deposition is associated with lower cognitive performance both in AD patients and in the normal elderly, but the association is modified by CR (education and intelligence quotient or education and occupation), suggesting that CR may be protective against amyloid-related cognitive impairment already at work in the preclinical phase of AD (Rentz et al., 2010; Garibotto et al., 2008). More recently, Ewers and colleagues (Ewers et al., 2013) observed in subjects with normal cognitive performance that higher education was associated with lower FDGPET in preclinical AD (CSF Aβ abnormal levels), but not normal elderly subjects, suggesting that cognitive reserve had a compensatory function to sustain cognitive ability in presence of early AD pathology. Another relevant protective factor for developing dementia with age is practicing physical exercise. The studies, mostly performed in cognitively intact adult or elderly subjects, reported benefits in tests of executive control, attention, verbal memory and verbal fluency in correlation with a higher fitness (Barnes et al., 2003). Higher levels of physical activity are associated with a larger superior frontal volumes and selectively moderated age-related medial temporal lobe. . 9 .

(10) atrophy (Bugg and Head, 2011). Recent studies examined whether physical activity is associated with incident cognitive impairment during follow-up with a community-based prospective cohort study. The results showed that compared with participants without physical activity, participants with moderate or high physical exercise levels at baseline significantly reduced risk of incident cognitive impairment after 2 years (Etgen et al., 2010); leisuretime physical activity at midlife was associated with a decreased risk of dementia and AD later in life especially among genetically susceptible individuals (Rovio et al., 2005; Larson et al. 2006). A meta-analysis of studies including patients with neurodegenerative diseases found that physical activity is inversely associated with risk of dementia: in particular, higher physical activity seems to be associated with a 28% reduction in incident dementia (Hamer and Chida, 2009). Frequent exercisers (>3/week) have been reported to have a stable or improved cognitive pattern over 5 years (Middleton et al., 2008). More recently, the already mentioned Yaffe et al. (Yaffe et al., 2009) study included, amongst the significant predictors of being a maintainer versus a minor decliner, engaging in weekly moderate to vigorous exercise. In their review on the effect of lifestyle activities on cognitive decline, Fratiglioni and colleagues concluded that social, mental and physical components of lifestyle share all the same pathways (Fratiglioni et al., 2004). From these findings, it has become evident that the stimulating activities can attenuate normal age-related cognitive changes and also reduce the risk for dementia in MCI.. . 10 .

(11) Figure 1: Model of the clinical trajectory of Alzheimer's disease (AD). The stage of preclinical AD precedes mild cognitive impairment (MCI) and encompasses the spectrum of presymptomatic autosomal dominant mutation carriers, asymptomatic biomarker-positive older individuals at risk for progression to MCI due to AD and AD dementia, as well as biomarker-positive individuals who have demonstrated subtle decline from their own baseline that exceeds that expected in typical aging, but would not yet meet criteria for MCI. Note that this diagram represents a hypothetical model for the pathological-clinical continuum of AD but does not imply that all individuals with biomarker evidence of AD-pathophysiological process will progress to the clinical phases of the illness (from Sperling et al., 2011). Figure 2: Hypothetical model of dynamic biomarkers in the pathophysiological sequence of AD including the preclinical phase. Aβ as identified by cerebrospinal fluid Aβ assay or PET amyloid imaging. Synaptic dysfunction evidenced by fluorodeoxyglucose (F18) positron emission tomography (FDG-PET) or functional magnetic resonance imaging (fMRI), with a dashed line to indicate that synaptic dysfunction may be detectable in carriers of the ε4 allele of the apolipoprotein E gene before detectable Aβ deposition. Neuronal injury is evidenced by cerebrospinal fluid tau or phospho-tau, brain structure is evidenced by structural magnetic resonance imaging. Biomarkers change from normal to maximally abnormal (y-axis) as a function of disease stage (x-axis). The temporal trajectory of two key indicators used to stage the disease clinically, cognitive and behavioral measures, and clinical function are also illustrated (from Sperling et al., 2011).. . 11 .

(12) MCI Diagnostic Criteria. MCI Subtype Classification. 1. Subjective cognitive complaint, preferably corroborated by an informant. MCI, amnestic, single domain. 2. Abnormal function for age in one or more domains of cognitive function. MCI, amnestic, multiple domains. 3. Evidence of decline in one or more aspects of cognitive function. MCI, non-amnestic, single domain. 4. Essentially normal functional activities. MCI, non-amnestic, multiple domains. 5. Absence of dementia Table 1: MCI Diagnostic Criteria and Subtype Classification (adapted from Nelson and O’Connor, 2008).. . 12 .

(13) COGNITIVE AND PHYSICAL STIMULATION: STUDIES IN HUMANS Primary care physicians are often asked to recommend lifestyle changes that might improve an older person’s chance for successful aging. The primary care physician can use insights from clinical, basic science, and pathological research to recommend intellectual, physical, and psychosocial stimulation as part of their cognitive wellness program for middle aged and older patients. Indeed, occupations with high mental demands may represent a form of mental exercise that supports brain function in older adulthood, as expressed by the ‘‘use it or lose it’’ hypothesis and may thus affect cognitive performance in older adults (Hultsch et al., 1999; Frick and Benoit, 2010). Successful aging as an optimal state implicates more than physical well being and fits the World Health Organization's definition of health as a state of complete physical, mental, and social well-being and not merely the absence of disease (Von Faber et al., 2001; Fig. 3). Participation in cognitively stimulating activities has been associated with reduced late-life cognitive decline in several studies (Bosma et al., 2002; Wilson et al., 2002; 2003; 2007). In particular, engagement in cognitively stimulating activities, such as reading, writing and playing games has been shown to contribute in delaying dementia’s onset (Verghese et al., 2003). The mechanism by which these activities exert protective effects is not yet clear. Recently, complex mental activity across the lifespan was correlated with reduced rate of hippocampal atrophy: that neuroprotection in medial temporal lobe may be one mechanism underlying the link between mental activity and lower rates of dementia observed in epidemiological studies (Valenzuela et al., 2008). The definition of the activities varies largely not only in the quantification used but also in the conceptual level of investigation: for example, some studies used simple quantitative assessment, such as number of social ties and time devoted to activities. Large variation is also present in the assessment of cognitive performance, ranging from very short global cognitive tests to large neuropsychological batteries testing multiple cognitive domains. With the. . 13 .

(14) available information, it is often not possible to identify the effect of a specific mental or physical activity. Furthermore numerous cognitive training interventions have been done in small-scale samples of subjects and without correct temporal relation between cause and effect (Fratiglioni et al., 2004). In normal elderly individuals, there are evidences that cognitive training helps to perform better in the specific tasks used for training (Valenzuela and Sachdev, 2006). But is not yet clear if the improvement can be transferred to untrained tasks and to real-world situations: this underlines the importance of the concept of enriched environment as driver of enhancement of neural plasticity and cognitive processes. A large randomized clinical trial (ACTIVE Advanced Cognitive Training for Independent and Vital Elderly), showed a durable beneficial effect from cognitive training on the targeted cognitive abilities at least 5 years after training, but poor effects on everyday function (Rebok et al., 2014). This supports the idea that cognitive training is a potentially efficient method to postpone cognitive decline in persons with mild cognitive impairment. But when we consider patients with MCI, to assess the benefits due to the intervention is even more difficult, since results from cognitive stimulating trainings involve heterogeneous groups. Most of the research on the effect of cognitive training in MCI has reported increased performance following training, documented by on objective measures of memory, whereas a minority reported no effect of training on objective cognitive measures. Interestingly, some of the studies that reported a positive effect of cognitive training in persons with MCI have observed large to moderate effect size. However, all of these studies have limited power and few have used long-term follow-ups or functional impact measures (Belleville, 2008). Therefore, the impact of non-pharmacologic cognitive interventions in elderly with MCI has yet to be elucidated. A randomised controlled trial showed that 14-sessions of cognitive training ran twice a week for 7 weeks significantly improved, with respect to the control group, the global cognitive status, measured by the Mini-Mental State. . 14 .

(15) Examination and the Alzheimer’s Disease Assessment Scale Cognition (Spector et al., 2003). Another beneficial effects of non-pharmacologic interventions on agerelated cognitive decline has been obtained with interventions based on physical exercise. The last few years have seen an increasing number of studies devoted to assess the effects of physical exercise on age-related decline via intervention approaches. Some of the first studies, which have followed this approach in nonpathological elders, observed that cognitive performance after an intervention of a few months was ameliorated in those subjects who performed physical training, in good correlation with the level of cardiovascular fitness and the pattern of brain activation with fMRI (Colcombe et al., 2004; Kramer and Erickson, 2007). A recent randomized controlled trial with 120 older adults showed that higher levels of physical activity enhanced spatial memory and increased the size of the anterior hippocampus; the increase hippocampal volume by 2% was associated with greater serum levels of BDNF (Erickson et al., 2011). In addition, Lautenschlager et al. (2008), in a study of adults over 50 with subjective memory impairment, found that a 6-month program of physical activity provided a modest improvement in cognition over an 18-month followup period. These results indicate that a higher level and variety of physical activity is associated with a lower cognitive decline and a lower risk for dementia. Which factors are most important in moderating this influence? A possible factor is the distinction between aerobic and non-aerobic physical activity. In a randomized controlled intervention, Kramer and colleagues (Kramer et al., 1999) observed substantial improvements on cognitive tasks requiring executive control in people who received aerobic training, compared with anaerobically trained people. This finding was confirmed in a subsequent meta-analysis (Colcombe and Kramer, 2003). Very few studies are available on the effects of training, cognitive or physical, on MCI or AD subjects and the efficacy of such interventions becomes less clear because of the lack of a certain diagnosis of degenerative disease in the. . 15 .

(16) population studied, as well as because of the inclusion of mixed samples (Ahlskog et al., 2011; Van Uffelen et al., 2008). In a recent meta-analysis of random controlled-trials of exercise effects on cognitive outcome in people with MCI, Gates and colleagues (Gates et al., 2013) observed that aerobic training was found to improve global cognitive function, verbal fluency, executive functions and memory. Questions remain regarding the magnitude, generalization, persistence, and mechanisms of benefits.. Figure 3: Quantitative model of successful aging (from Von Faber et al., 2001).. . 16 .

(17) ENRICHED ENVIRONMENT: ANIMAL STUDIES As introduced above, Environmental enrichment (EE) is an experimental protocol classically defined as “a combination of complex inanimate and social stimulation” (Rosenzweig et al., 1978) and which provides animals with the opportunity to attain high levels of voluntary physical activity on running wheels and to enhance exploration, cognitive activity, and social interaction (Fig.4). Animals reared under enriched conditions are housed in large groups, in wide cages where a variety of different and colorful objects (e.g. running wheels, platforms, boxes, toys, tunnels, shelters, stairs and nesting materials) are placed and changed frequently. Thus, EE definition is based on the comparison with alternative rearing conditions, such as the standard condition (SC), in which the animals are reared in small social groups and in very simple cages where only nesting material, food and water are present. Several studies showed that the morphology, chemistry and physiology of the brain can be altered by modifying the quality and intensity of environmental stimulation, with EE eliciting remarkable plastic responses ranging from molecular to anatomical and functional changes. EE can indeed be used as a noninvasive strategy to modulate brain plasticity throughout life; EE can accelerate the development of the central nervous system (Sale et al., 2004; Landi et al., 2007; Sale et al., 2007a), and can reopen plasticity windows in the adult cortex (Sale et al., 2007b; Baroncelli et al., 2012). A large number of studies highlighted the fact that EE modifies the behaviour of animals, leading to a strong improvement in complex cognitive functions, particularly learning and memory (van Praag et al., 1999; Duffy et al., 2001; Bennett et al., 2006). Rodents living in EE conditions display increased levels of hippocampal long-term potentiation (LTP), a physiological model of synaptic plasticity related to learning and memory (van Praag et al., 2000). This functional improvement is accompanied by prominent changes at the anatomical level, with robust increments in cortical thickness and weight (Rosenzweig et al., 1964; Beaulieu et al., 1987) and modifications of neuronal morphology, in terms of increased dendritic arborization, number of dendritic . 17 .

(18) spines, synaptic density and postsynaptic thickening, occurring in several regions of the brain (Holloway, 1966; Kozorovitskiy, 2005; Mohammed et al., 2002). Moreover, studies have reported that EE reduces apoptotic cell death (Young, 1999), and increases hippocampal neurogenesis (Kempermann et al., 1997). At the molecular level, EE causes a significant change in the expression of a large set of genes involved in neuronal structure, excitability, synaptic transmission and plasticity (Rampon et al., 2000), modulating the synthesis and secretion of neurotrophic factors throughout the brain (Ickes et al., 2000; Pham et al., 2002) and affecting the cholinergic, serotoninergic and noradrenergic systems (Rosenzweig et al., 1967; Rasmuson et al., 1998; Naka et al., 2002). Moreover, rearing animal models of nervous system disorders, including neurodegenerative diseases (Nithianantharajah and Hannan, 2006; Baroncelli et al., 2010) in an enriched environment, leads to striking beneficial effects, for example in delaying the progression and/or in ameliorating the symptoms of neurological disorders. Studies of environmental effects on age-related changes in cognitive function widely indicated that living in complex, stimulating environments (both from youth and only during old age) rescues cognitive deficits or, at least, ameliorates the performance on tests of learning and memory in aged animals (Doty, 1972; Cummins et al., 1973; Warren et al., 1982; Winocur, 1998; Soffié et al., 1999; Kempermann et al., 2002; Kobayashi et al., 2002; Frick and Fernandez, 2003; Bennett et al., 2006; Leal-Galicia et al., 2008). One group of factors particularly sensitive to environmental stimuli and exerting potent functions in the nervous system are the neurotrophic factors, or neurotrophins, a class of secreted proteins promoting neuronal development and survival, which include NGF (Nerve Growth Factor) and BDNF (BrainDerived Neurotrophic Factor). Neurotrophic factors are strongly implicated in regulating structural and functional neural rearrangements to sensory stimulation (Bonhoeffer, 1996; Berardi et al., 1999; Thoenen, 2000), thanks to their capacity to act as ideal players following the classic Hebb’s principle of activitydependent plasticity of the nervous system.. . 18 .

(19) In addition to neurotrophins, sustained levels of physical exercise occurring either in EE or as an individual component of environmental stimulation can increase brain uptake of other physiologically relevant trophic factors, such as IGF-I (Insuline-like Growth Factor), which mediates most of the known effects of exercise on the brain, including increased BDNF expression and c-Fos activation, increased hippocampal neurogenesis and protective effects against brain insults (Carro et al., 2000; 2001; Trejo et al., 2001; Koopmans et al., 2006); interestingly, IGF-1 favors clearance of Aβ from the brain (Carro et al., 2002). Recently, EE has been also linked with chromatin modifications through increased histone-tail acetylation (Fischer et al., 2007).. Motor. Visual. Cognitive. Somatosensory. to adulthood (oft age in rodents), t the developing b adult brain. Enri weaning in rode effects, such as al. Environmental Environmental e wild-type mice a to behavioural. A investigating the that enrichmen ness11–13. Subsequ enrichment incr the number of d apses on some ne Figure 1 | Environmental enrichment and the effects of enhanced sensory, enrichment incr cognitive and motor stimulation on different brain areas. Enrichment can promote neuronal activation, signalling and plasticity throughout various brain regions. Enhanced the integration of Figure 4: Environmental Enrichment (EE). Enrichment can promote neuronal activation, sensoryand stimulation, somatosensory and visual input, activates theincluding signalling plasticity including throughoutincreased various brain regions. Enhanced sensory stimulation, circuits9,22–26. This somatosensory (red)and andvisual visualinput, (orange) cortices. Increased cognitive stimulation — forcortices. increased somatosensory activates the somatosensory (red) and visual (orange) gested to be med Increased cognitive stimulation — for example, the to encoding information relating to spatial maps, example, the encoding of information relating spatial of maps, object recognition, vascular endothe object recognition, novelty andofmodulation —activate is likely the to activate the hippocampus (blue) and novelty and modulation attentionof —attention is likely to hippocampus (blue) and recruitment of T other cortical areas. In addition, enhanced motor activity, such as naturalistic exploratory movements other cortical areas. In addition, enhanced motor activity, such as naturalistic exploratory (including fine motor skills that differ radically from wheel running alone), stimulates areas such as the Many of thes movements (including fine motor skills that differ radically from wheel running alone), motor cortex and cerebellum (green) (from Nithianantharajah and Hannan, 2006). with enrichment stimulates areas such as the motor cortex and cerebellum (green). of genes involved ticity29. Enrichme experimental methods. Enrichment objects generally such as brain-der vary in composition, shape, size, texture, smell and colour nerve growth fac (although diurnal activity patterns and the limitations of neuronal signalli the rodent visual system could mean that somatosensory expression of syn and olfactory stimuli are the most salient). In addition, vesicle protein syn there is variation in whether enrichment involves access 19 protein (PSD-95) ( to running wheels, which has significant implications induced enhance as enhanced voluntary exercise alone has effects on the togenesis. Furthe.

(20) MEMORY FORMATION AND CONSOLIDATION A major breakthrough in understanding how the brain accomplishes the task of learning and memorizing began with the study of a person known by his initials, H.M. (Scoville and Milner, 1957). As a child, H.M. developed a severe, difficult-to-treat form of epilepsy. When traditional therapies failed to help, H.M. underwent an experimental surgical treatment: the removal of the medial regions of his temporal lobes. The surgery worked in that it greatly alleviated the seizures, but it left H.M. with severe amnesia. He could remember recent events for only a few minutes and was unable to form explicit memories of new experiences. Despite his inability to remember new information, H.M. remembered his childhood very well. From these unexpected observations, researchers concluded that the parts of H.M.’s medial temporal lobe that were removed, including the hippocampus and parahippocampal region, played critical roles in converting short-term memories to long-term, permanent ones. Because H.M. retained memories of events that occurred long before his surgery, it appeared that the medial temporal region was not the site of permanent storage but instead played a critical role in the initial processing, organization and storage of these memories, while permanent storage of memories was accomplished elsewhere in the brain. Since then, scientists have learned that the medial temporal region is closely connected to a widespread network of cerebral cortex areas and it has been suggested that whereas the medial temporal lobe is important for forming, organizing, consolidating and retrieving recently formed declarative memories, it is the cortical areas that are important for long-term storage of detailed knowledge about facts and events and how this knowledge is used in everyday situations (Moscovitch et al., 2005). In particular, cortical areas in the prefrontal region have been suggested to be crucially involved in long term storage of declarative memories. According to this new model of declarative memory systems, declarative memories depend initially on the medial temporal lobe system, including the hippocampus, but, as these memories mature, they become increasingly. . 20 .

(21) dependent on other brain regions such as the cortex (Fig. 5). Therefore, after the initial formation and consolidation in the hippocampus, it is hypothesised that memory traces are gradually transferred or copied into neocortical areas, until, for remote memories, only the latter are involved in storage and recall. This process of gradual recruitment with time of cortical areas in storage and recall of declarative memories is called memory system consolidation (Frankland and Bontempi, 2005) This system consolidation is a prolonged process and involves gradual reorganization of brain regions supporting memory storage and recall. According to the standard consolidation model (Squire et al., 1995; McClelland et al., 1995), experience is initially encoded in parallel in hippocampal and cortical networks, and subsequent reactivation of the hippocampal network reinstates activity in different cortical networks. This coordinated replay across hippocampal–cortical networks leads to gradual strengthening of cortico-cortical connections, which eventually allows new memories to become independent of the hippocampus and to be gradually integrated with pre-existing cortical memories (Fig. 6). In an alternative view, the multiple trace theory proposes that, although experience is initially encoded in distributed hippocampal–cortical networks, the hippocampus is always required for rich contextual or spatial detail (Nadel and Moscovitch, 1997; Moscovitch et al., 2005). The assumption that reactivation of memories initiates a process of reorganization is present in both models: however, standard models predict that reorganization occurs in cortical networks, while multiple trace theory predicts that reactivation should also lead to the generation of new traces within the hippocampus.. . 21 .

(22) cambiamenti sono rapidi e transienti. Una successiva riattivazione della rete ippocampo-corteccia porta ad un graduale rafforzamento delle connessioni cortico-corticali e alla creazione di nuove. Il rafforzamento incrementale di queste. tical memories, once the hipconnessioni permette volved in this process.. ai nuovi ricordi di diventare indipendenti dall’ippocampo e. di essere progressivamente integrati con le memorie pre-esistenti. Queste. eocortex memorie corticali diventano più resistenti al tempo e alle interferenze rispetto a n studies suggest that hippocent memories more severely quelle ippocampali. La caratteristica fondamentale di questo modello è che i consistent with the idea that me-limited role in the storage cambiamenti y. However, these studies did della forza tra le connessioni del sistema ippocampale e delle memories age and the hippoFigure 5: Figure Activation of rapidi the hippocampus and Neocortex neocortex bylerecent and remote aree e transitori, mentre modifiche delle 2. sono Activation of the Hippocampus by Recent aged, specificdiverse neocortical re- corticali and Remote Memories memories. Recent and remote memories differentially activate the hippocampus and neocortex. As diate access to remote memomemories all’interno age, the hippocampus becomes disengaged. This islente evidenced byhippocamdecreasesnel in activity following connessioni areememories corticali sono e the resistenti tempo (Fig.5). Recentdelle and remote differentially activate compelling evidence for this remote memory retrieval and the inability of lesionsage, to affect performance. becomes In contrast, neocortical areas pus and neocortex. As memories the hippocampus ontempi et al., 1999; become Takehara more engaged as memories This is evidenced by increases in activity following retrieval and disengaged. This age. is evidenced by decreases in activity following al., 2004). the emergence of lesion effects on performance Wiltgen et al., 2004). remote memory retrieval and (from the inability of lesions to affect perforained animals on a hippocammance. In contrast, neocortical areas become more engaged as memories age. This is evidenced by increases in activity following rning task and then monitored retrieval and the emergence of lesion effects on performance. deoxyglucose uptake] followent or remote memories. Remories produced more robust However, retrieval of remote context memories proan remote memories, a result duced the opposite effect: reduced IEG expression in el that proposes progressive the hippocampus and increased expression in neocortippocampus during memory cal areas. The functional importance of this activation t, several neocortical areas was demonstrated by blocking activity in the anterior , showed the opposite pattern cingulate cortex (ACC) during memory tests. It was ctivation during remote memfound that inactivation of this PFC structure impaired e compelling evidence in favor retrieval of remote but not recent context fear memories. cal networks in remote memThis suggests that while the hippocampus is engaged, , for the first time, that specific context fear memories do not require the PFC, but with reorganization of neuronal in the system consolidation ct become more engagedFigure as 6: Time-dependent the progressive disengagement of theactivation hippocampus, process of memory traces. Declarative memories initially depend on the medial temporal lobe system, e. the PFCStandard becomes di essential for the consolidation, storFig. 5hippocampus Modello Consolidamento (Bontempi et al., 2005). including the (HPC), but, as these memories mature, they become increasingly dependent on were also the first to point to age, or retrieval of these memories. other brain regions such as the prefrontal cortex (PFC). This system consolidation is a prolonged process cortex that become activated In a similar fashion, a recent paper by Maviel et al. and involves gradual reorganization of the brain regions that support memory (from Frankland and tion. The data revealed that2005). (2004) increased IEG expression neocortical Non viene messo in found discussione l’esistenza di unin consolidamento rapido da Bontempi, m recent to remote the PFC regions following the retrieval of remote but not recent l, and temporal cortex all bespatial memories in mice. inactivation of the dettagli sulla sua parte dell’ippocampo, anzi ad oggi siamoTargeted a conoscenza di molti allowed researchers to examACC or prelimbic cortex impaired the retrieval of remote sses by targeting arbutsembra not recent spatial memory. Consistent with similar basespecific molecolare che essere simile tra le specie (Kandel, 2001). dies employing this strategy findings by Frankland et al. (2004), this study also reFC are critical forQuesta the consolithat suggests synaptic structural teoria è vealed basata evidence su un modello proposto da Squire forte delle evidenze pendent memories (Takehara changes take place in the neocortex during remote al., 2004; Maviel et al., 2004).sia damemory Animals testedretrograda 30 days after provenienti casi diconsolidation. pazienti con amnesia (Teng et al., 1999; al. (2003) showed that hippotraining showed increases in the expression of the se a large deficit in trace con-et al., 2000), growth-associated protein GAP43 to a 1 daye genetici. Rosenbaum sia su studi animali di compared lesione, imaging y but not late after training. retention group. This suggests that synaptic changes in e medial PFC (including the cortical regions may underlie the formation or stabilizagulate cortex) produced the tion of remote memories in15the cortex. A related finding d no effect early after training is that mice with a dominant-negative transgenic PAK, en made at later time points. a regulator of actin remodeling, also exhibit remote Figure 7: Areas of the brain significantly involved in declarative memory, shows in a midline tempi’s activation data (1999) memory impairments (Hayashi et al., 2004). The domiview of the brain. The medial temporal lobes, including the hippocampus and parahippocampal cortex, nce that activation ofarePFC renant-negative transgene changesas in critical for normal declarative, andPAK in particular, spatialproduced memory functioning, are other brain regions s to remote memories. plasticity and spine morphology in the cortex but not highlighted in blue (adapted from Budson and Price, 2005). ound increases in neocortical the hippocampus. These changes did not affect the ac) expression following the requisition or retention of recent spatial memories, as mice fear memories in mice. IEGs exhibited normal performance 1 day after learning. How 22 of neuronal activation, and ever, the changes in synaptic morphology did affect h as Zif268, are also required the long-term stability of spatial memories. In contrast, n and memory (Vann et al., contextual memories were already affected 1 day after.

(23) Spatial memory is a type of declarative type of memory and is widely used to investigate memory formation, consolidation and recall in animal models. Experiments in rodent models employing lesion, electrophysiological, genetic, and neuroimaging approaches, have long established that the hippocampus plays an essential role in the formation of spatial memories (O’Keefe and Nadel, 1978; Burgess et al., 2002; Morris et al., 2003; Nakazawa et al., 2004; Leutgeb et al., 2005). As far as the system consolidation is concerned, it has been shown that spatial memory undergoes system consolidation and become additionally dependent on a broadly distributed cortical network. Many studies pointed out that the medial prefrontal cortex (mPFC) might have a privileged role in processing remote spatial memory (Bontempi et al., 1999; Maviel et al., 2004; Frankland et al., 2004; Teixeira et al., 2006). In both humans and rodents the mPFC consists of several highly interconnected regions, including the anterior cingulate cortex, and prelimbic and infralimbic cortices (Fig. 7), and these regions are reciprocally connected to sensory, motor, and limbic cortices (Uylings et al., 2003). In addition, several experiments have provided evidence that inactivation or lesions of the prelimbic or anterior cingulate cortices block recall of remote memory, but not recent memories (Maviel et al., 2004; Frankland et al., 2004), even in the presence of an intact hippocampus (Fig. 8). Although formation and stabilization of long-lasting associative memories require time-dependent coordinated hippocampal-cortical interactions, the underlying mechanisms remain unclear. One possibility is the involvement of hippocampal neurogenesis. It has been shown that new neurons generated in the dentate gyrus become functionally integrated into existing neural circuits (Deng et al., 2010); during the spatial training, newly generated neurons, which are more susceptible to induction of activity dependent plasticity, would be included in the spatial memory trace in the hippocampus and would be subsequently recruited upon remote memory retrieval (Trouche et al., 2009; Arruda-Carvalho et al., 2011). Thus, these adult-generated neurons become tagged in order that, once mature, they are preferentially recruited into hippocampal networks underlying remote. . 23 .

(24) spatial memory representation, and they could act as time clusters for the storage of long-term episodic memories. Therefore tampering with the level of hippocampal neurogenesis could interfere in the hippocampus-dependent period of memory. It has been demonstrated that a decreased neurogenesis is accompanied by a prolonged hippocampus-dependent period of associative memory, and conversely, enhanced neurogenesis by voluntary running-wheel exercise sped up the decay rate of this dependency (Kitamura et al., 2009). Another possibility, supported by many experimental data, is that during system consolidation there is a progressive strengthening of cortico-cortical connections through activity dependent synaptic plasticity mechanisms. According to this model, cortical synaptic plasticity mechanism would be critical in allowing memories to migrate from the hippocampus to the cortex. Indeed, studies in genetically mutant mice for α-CaMKII (Frankland et al., 2001) and p21 activated kinase (Hayashi et al., 2004) showed that these mice, which have normal hippocampal but abnormal cortical plasticity, were unable to form enduring, hippocampus-independent memories (Fig. 9). Morphological changes at synaptic sites is a necessary step towards long lasting changes in synaptic efficacy underlying long term synaptic plasticity: as a further sign of cortical synaptic plasticity being involved in system consolidation, it has been demonstrated that cortical consolidation involves morphological synaptic plasticity, mediated by the expression of proteins involved in axonal growth and sprouting (Frankland et al., 2004; Maviel et al., 2004). In addition, timedependent changes in spine density in hippocampal and cortical networks have been shown to take place during the formation of recent and remote memory (Restivo et al., 2009; Vetere et al., 2011). Also associative olfactory memory is a type of hippocampus dependent, declarative type of memory and it refers to the association of an odor with other odors or with non-olfactory sensory stimuli. In animals, memory for environmental odors plays a vital role because it regulates many behaviors that are crucial for survival. Highly emotional or at least particularly ethologically relevant olfactory learning occur during an animal’s major life events. Without a properly functioning of this memory, animals could be at risk of dangerous. . 24 .

(25) stimuli in its environment such as the predator’s odor or adverse stimuli. In particular, association of a food odor with the smell of a conspecific breath is linked to food choices (Valsecchi et al., 1996). Also in this case there is a system consolidation. Lesburguères and colleagues (Lesburguères et al., 2011), to pinpoint the post-learning mechanisms underlying the hippocampal-cortical dialogue during the course of systems-level memory consolidation, used the social transmission of food preference (STFP) paradigm, which involves an ethologically based form of associative olfactory memory. Cellular imaging of the activity-dependent gene c-Fos coupled to region-specific pharmacological inactivation by using tetrodotoxin or the AMPA receptor antagonist revealed a necessary role played by the HPC in acquisition of associative olfactory memory, a transitory role of HPC in remote memory storage of associative olfactory information and a concomitant increase in Fos immunoreactivity in the OFC in remote memory storage of associative olfactory information. Accordingly, remote memory retrieval examined at day 30 was impaired when hippocampal activity was silenced during the early, but not the late, post-learning period. Alternatively, disrupting cortical activity during the late phase of the consolidation process may. have. interfered. with. neuronal. mechanisms. underlying. memory. maintenance. The study evidenced that neurons in the rat cortex must undergo an early “tagging process” upon encoding to ensure the progressive hippocampal-driven rewiring of cortical networks that supports remote memory storage. Indeed, the preserved memory at day 15 after partial OFC inactivation was associated with an unusual maintenance of hippocampal activation at this time point, possibly to compensate for the absence of gradual recruitment of the OFC normally seen in control animals over the 30-day period. HPC can be act as a temporary memory buffer, but the alteration of cortical tagging led to an unstable memory trace: this indicates that synaptic tags may serve as an early and persistent signature of activity in the cortex that is necessary to ensure the progressive rewiring of cortical networks that support remote memory storage. The identity of synaptic tags needs to be further explored, although potential candidates could be AMPA, N-methyl-D-aspartate receptor–dependent. . 25 .

(26) REMOTE. *. REMOTE. Zif268 counts (% cont. icantly lower than that of paired contro subjects (Fig. 2A), which raises the possi bility that inhibitory influences may ulti 100 mately control the level of engagement o the hippocampal formation in memory con *† 50 solidation (7). A similar decrease was als 0 observed in the posterior cingulate corte Day 30 Day 1 [Fig. 1A; F(1,36) ! 6.70, P " 0.05], whic suggests a conjoint involvement of hip Fig. 2. Time-limited role of hippocampus in remote spatial memory storage and retrieval. (A) Zif268 counts relative to controls in dorsal (top) and ventral (bottom) hippocampus after testing pocampal and certain cortical network for recent (day 1) versus remote (day 30) memory retention. Zif268 expression was elevated in during early memory processing (7). Hip these structures after testing recent memory but, in contrast, was decreased below control levels pocampal disengagement was specificall after testing remote memory. (B) Photomicrographs of Zif268 labeling in dorsal hippocampus related to memory consolidation, sinc (CA1d) after testing for spatial reference memory, recent (top) as compared to remote (bottom). mice tested over the same time period in (C) Hippocampal disengagement, shown in (B), was not observed in animals tested for working working memory paradigm in which infor memory. *P " 0.01 versus respective controls (100% line); †P " 0.01 versus day 1; n ! 10 mice per group. Scale bars, 50 $m. mation changes from trial to trial (and i thus stored only temporarily) did not show decreased Zif268 expression in the hip pocampus [Fig. 2C and fig. S5, memor type # time interaction: F(1,34) ! 31.67 P " 0.001]. We next infused the anesthetic lidocain into selected brain regions before testin for memory retention to transiently silenc neuronal activity and thereby to minimiz any possible compensatory mechanism within memory systems associated wit irreversible lesions (13). Inactivation o hippocampus or posterior cingulate corte disrupted recent, but not remote, memor retrieval (Fig. 3, A and B; fig. S6), whic indicated that these two interconnecte brain regions (19) mediate information pro cessing in parallel during early stages o memory consolidation (7). In contrast, si lencing neuronal activity in prefrontal o anterior cingulate cortex selectively dis rupted retrieval of remote memories (Fig Fig. 3. (A) Effects of neuronal inactivation of the prefrontal, anterior or posterior cingulate 3, A and B; fig. S6). These findings provid cortices, or dorsal hippocampus by lidocaine (black bars) as compared with vehicle [artificial Figure 8: Effects of neuronal inactivation of the prefrontal, anterior or posterior cingulate evidence for a critical requirement for spe cerebrospinal fluid (aCSF), white bars] on recent (day 1) and remote (day 30) memory retrieval. neocortical cortices, or dorsal hippocampus. (A) Inactivation by lidocaine (black bars) comparedcific with vehicleregions in remote spatia (B) Coronal diagrams [adapted from (30)] showing the location of injection sitesas in prefrontal memory retrieval and indicate that the hip (top) and dorsal hippocampus (CA1d, bottom) plotted onto a single plane (AP %1.7 mm (artificial cerebrospinalcortex fluid, aCSF, white Efficacy bars) ofonlidocaine recentinactivation (day 1)was and remote (day the 30)area memory retrieval. and –2.0 mm, respectively). verified by assessing pocampus does not play a permanent rol blockade Zif268 staining. Injection sites for subjects receiving vehicle (open as longdorsal as memories remain viable (6–8 (B) Coronal diagramsexhibiting showing the oflocation of injection sites in prefrontal cortex (top)for and triangles) are plotted on the left; the injection sites (black triangles) and maximum extent 20). They are consistent with two observa hippocampus (CA1d, (shaded bottom) plotted onto a single plane (AP +1,7 mm and –2,0 mm, respectively). blue area) of lidocaine inactivation are shown on the right of each section. Animals tions: that functional disruption of cortica included in was the study only if by cannula tips were the correctly within targeted Efficacy of lidocaine were inactivation verified assessing arealocated exhibiting blockade of Zif268 plasticity preferentially disrupts the estab structures and complete IEG inactivation was circumscribed to the region of interest with of enduring memories in CaMKI diffusion spread along the guide cannula *P " 0.05 **P " 0.01 versus staining. Injection sitesminimal for subjects receiving vehicle (open track. triangles) areandplotted on the left;lishment the injection vehicle-injected controls; n ! 7 to 10 mice per group. Scale bar, 100 $m. knockout mice (21, 22) and that in semanti 150. and the mechanisms involved in control of gene transcription, such as histone. acetylation, involved in chromatin remodelling events (Lesburguères et al., 2011).. sites (black triangles) and maximum extent (shaded blue area) of lidocaine inactivation are shown on the right of each section.98 Animals were included in the study cannula tips were correctly located within 2 JULYonly 2004 ifVOL 305 SCIENCE www.sciencemag.org Cortical Synaptic Morphology and Long-Term Memory targeted structures and complete inducible immediate early gene inactivation was circumscribed to the 781 region of interest with minimal diffusion spread along the guide cannula track. *p <0,05 and **p <0,01 versus vehicle-injected controls; n =7 to 10 mice per group. Scale bar, 100 µm (from Maviel et al., 2004).. Figure 6. Normal Acquisition and Impaired Consolidation/Retention of Spatial and Contextual Fear Memories in dnPAK Transgenic Mice (A) Normal acquisition of spatial memory in the hidden-platform Morris water maze. The decreases in escape latency (left panel) and path length (right panel) were equivalent between the wild-type (open circle) and transgenic mice (filled circle; n ! 34 each, p " 0.05, repeated-measures ANOVA). (B) Results of probe trials given on day 14 (end of training, left), day 15 (1 day after the completion of training, middle), and day 35 (21 days after the completion of training, right). (Upper panel) Mean percent of time spent searching in each quadrant. Dotted lines depict chance level (25%) for random searching. (Lower panel) Mean number of platform crossings. There were significant group differences in both measures on day 35 but not on days 14 and 15. *p # 0.05; **p # 0.01. T, target quadrant; L, adjacent left quadrant; R, adjacent right quadrant; O, opposite quadrant. (C) Freezing responses for the wild-type and transgenic mice in fear conditioning. The transgenic mice showed a significantly impaired context-associated fear memory at 24 hr but not at 40 min or 7 hr after conditioning. At 48 hr after conditioning, the transgenic mice exhibited a comparable freezing response to a novel (unconditioned) context (“pre-tone” period) as well as an intact toneassociated fear memory relative to wild-type mice. *p # 0.03.. Figure 9: Normal Acquisition and Impaired Consolidation/Retention of Spatial and Contextual Fear Memories in dnPAK Transgenic Mice. (A) Normal acquisition of spatial memory in the hidden-platform Morris water maze: the decreases in escape latency (left panel) and path length (right panel) were equivalent between the wild-type (open circle) and transgenic mice (filled circle; n =34 each, p >0,05, repeated-measures ANOVA). (B) Results of probe trials given on day 14 (end of training, left), day 15 (1 day after the completion of training, middle), and day 35 (21 days after the completion of training, right): mean percent of time spent searching in each quadrant. There were significant group differences in both measures on day 35 but not on days 14 and 15. Dotted lines depict chance level (25%) for random searching. **p <0,01. T, target quadrant; L, adjacent left quadrant; R, adjacent right quadrant; O, opposite quadrant (From Hayashi et al., 2004).. . 26 ity (enhanced LTP and reduced LTD). In contrast, spine morphology and synaptic plasticity in the hippocampus were unaltered in dnPAK transgenic mice. Notably, dnPAK transgenic mice also displayed specific im-. cortex of dnPAK transgenic mice could in part reflect PAK’s role in developmental spinogenesis. However, we believe that the contribution of developmental effects should be minimal, since endogenous PAK activity is.

(27) AIM OF THE THESIS . The aims of the thesis are: •. to study the effects of a physical and cognitive training program lasting 7. months in subjects with MCI, using a neuropsychological battery to assess the effects of the intervention at the end of the 7 months and in subsequent followup tests; •. to study the effects of EE on spatial memory system consolidation process. in adult mice; •. to study the effects of EE on declarative memory system consolidation. process in aged mice and to investigate the underlying mechanisms. In the first project, I have assessed the efficacy of an “enriched environment” approach in aged MCI subjects participating in the “Train the Brain” study.. Train The Brain is a clinical and experimental study on the. effectiveness of cognitive and physical training in slowing progression towards dementia, and on the relationship between mental and cardiovascular fitness. As outlined in the Introduction, cognitive decline due to ageing is becoming a major clinical and economic issue. Italy has more than 700,000 patients with dementia and 100,000 new cases are detected every year. No treatment is currently available for dementia: drugs currently used are poorly effective and do not prevent, heal, stop, or delay the progression of the disease. It is therefore crucial to find interventions and lifestyle factors to counteract and slow cognitive decline from an early stage. In the last years several epidemiological studies on human beings, supported by many experimental studies on animal models, have shown that participation in cognitively stimulating activities, such as reading, writing and playing games, and practicing physical exercise has a positive effect on brain function in the elderly and has been associated with reduced late-life cognitive decline. (Wilson et al., 2007; Laurin et. al, 2001; Fratiglioni et al., 2004; Kramer and Erickson, 2007). On the other hand, controlled intervention studies on human beings are few and show methodological limitations.. . 27 .

(28) Train the Brain study has focused on the possibility that a combination of cognitive and physical training in the prodromic stages of dementia might prevent clinic onset of dementia or slow disease progression. Such a prodromic stage is the MCI condition (for diagnostic criteria see Winblad et al, 2004; Portet et al, 2006). It is now established that the pathophysiological process of dementia begins many years before the clinical diagnosis of dementia. MCI dramatically raises the risk of developing dementia: MCI subjects have a much higher probability of evolving in dementia subjects in comparison with individuals of the same age but without MCI. In this prodromic condition, a progressive decrease of the number and quality of synaptic connections in specific areas of the brain has been hypothesized; interventions aimed at counteracting the decrease in synaptic function and density, augmenting neural plasticity and the development and sparing of mnemonical circuits, could slow the progression to overt dementia. Then, an early detection of cognitive impairment could allow to exploit. the. residual. plasticity. of. the. nervous. system. through. therapeutic/rehabilitative interventions. The working hypothesis for this project is that physical and cognitive stimulation could positively affect cognitive decline in subjects at risk for dementia or with dementia at its early stage. This could delay the loss of selfsufficiency, therefore improving quality of life both for the patient and his/her caregivers with a safe and relatively cheap intervention, also reducing direct and indirect costs for caregiving for the families and the National Health Service. The present study is interventional, with random allocation of subjects to the intervention or the control group. The projects phases are: •. Screening, cognitive baseline evaluation (T0), allocation to intervention or. control group; •. Baseline evaluation of brain volumetry and function (T0);. •. Baseline cardiovascular assessment (T0);. •. Intervention for the intervention group, usual care for the control group;. •. Cognitive Evaluation at the end of intervention (or after 7 months usual. care), T7, and 12 months after intervention completion or usual care (T19).. . 28 .

(29) •. Cardiovascular assessment and evaluation of brain volumetry and. function (T7). The results obtained at T7 indicate that the intervention causes an improvement in cognitive performance. Data at T19 are not yet available. To investigate the mechanisms of action of environmental enrichment in promoting cognitive performance in aged subjects I performed experiments in animal models, concentrating on EE effects on system consolidation. In a first experiments in animals, I tested whether exposure to EE affects memory system consolidation in adult mice. In particular, I have investigated whether EE can accelerate the involvement of the neocortex characterizing the time-dependent reorganization of cortical area activation upon memory recall in a brain-wide manner, not restricting my analysis only to hippocampus and mPFC. To obtain a time course of neocortical recruitment I evaluated cortical activation at different intermediate temporal points following learning, instead of employing only a recent (1 day) and remote (30 days) retention interval, in order to determine whether different neocortical areas are involved at different time points. Wild type mice, housed in Standard or in Enriched Condition, were subjected to a spatial learning (Morris water maze) and then tested 1, 10, 20, 30 and 50 days after learning to evaluate system consolidation process. Mice were sacrificed following probe tests and the regional expression of the inducible immediate early gene c-Fos was mapped, as an indicator of neuronal activity (Hall et al., 2001; Fleischmann et al., 2003). I found that EE induces an early recruitment of mPFC, the final storage site in the cortex, and that EE induces an involvement of distributed cortical network, that seems not to be engaged in recall of trace memory in standard housed mice. In the second experiment in animals I tested whether EE could affect system memory consolidation in aged mice using the social transmission of food preference (STFP) test (Galef et al., 2003; Wrenn, 2004; Wrenn et al., 2003; Valsecchi and Galef 1989). In this task, mice learn, within only one single interaction session of 30 min, about the safety of potential food sources by. . 29 .

(30) sampling the odour of those sources on the breath of littermates. The acquired olfactory memory is robust and long-lasting, which makes the STFP task particularly suitable for studying the processes underlying remote memory formation and the final memory destination area, the orbitofrontal cortex (OFC). I trained in the STFP aged mice, either left in standard cages or put for 40 days in EE and tested them for memory retrieval either 1 day (recent memory) or 30 days (remote memory) later. Mice were sacrificed following probe tests to analyse the activity-dependent gene c-Fos in the hippocampal formation (HPC) and OFC. Only EE mice successfully performed the recall task and showed an increased activation of OFC in the remote memory recall. Finally, I proceeded to verify the presence of early histone acetylation in OFC 1 hour after learning: there is an increase in histone H3 acetylation in EE but not in non EE animals, suggesting a failure in early OFC tagging which is rescued by EE.. . 30 .

(31) MATERIALS AND METHODS. . TRAIN THE BRAIN. . Train the Brain - Study protocol Symptoms are identified with an advanced set of diagnostic tests. In perspective, the aim is to develop a non-pharmacologic therapeutic strategy, which could be easily applied in clinical practice by structures of the National Health Service. When the study is completed, it will be possible to know if the program of physical exercise and cognitive stimulation has been able to reduce, to a statistically significant degree, the progression of cognitive decline and of brain damage in subjects with MCI, as measured, respectively, with neuropsychological tests and with morpho-functional techniques. If this were to be the case, the combined physical/cognitive intervention procedure will be proposed as non-pharmacological preventive and therapeutic strategy (Tab. 2). A final follow-up evaluation, 12 months after the end of the intervention, will provide information about the duration of the effects. The eligible population for the study includes elderly (65-89 years) subjects with Mild Cognitive Impairment confirmed at the neurological examination; severe neurological pathologies such as brain lesions or conditions barring participation to the cognitive or physical training program is the only substantial exclusion criteria. Phase 1 - Patient recruitment and baseline cognitive evaluation: a first screening with Mini-Mental State Examination (MMSE), Clock drawing test and CDR has been performed to select potentially eligible subjects from the general population, with the contribution of General Practitioners. These potentially eligible subjects underwent a clinical confirmation with a standard set of neuropsychological tests, which also provide the baseline cognitive status, and a comprehensive collection of medical history and physical, cognitive and affective examination; subjects with a confirmed diagnosis of MCI and matching inclusion criteria have been enrolled in the study and randomly assigned either. . 31 .