Effects of aging on the hippocampal formation in a naturally occurring animal model of mild cognitive impairment

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Abstract

Vertical integration is being used to great advantage in neurobiological research on the basis of age-related cognitive decline. Such research bridges analysis between the molecular and cellular levels and the outcome of impaired psychological functions. Current use of animals models within this paradigm has defined mild cognitive impairment in a subpopulation of outbred aged Long-Evans rats by assessment of hippocampal-dependent spatial cognition. Aged rats with cognitive impairment exhibited no loss of neurons in the hippocampus. Current research is focused on the functional alterations in neurons by methods which assess transcriptional mechanisms and signaling pathways.

Introduction

A major shift in demographics occurred in the 20th century as average life expectancy lengthened from 40 to 50 years of age in the early 1900s to approaching 80 years of age at the end of the last century. While medical progress has made it possible for succeeding generations to live longer, it has become clear that even if the aging brain does not succumb to major neurodegenerative disease, discernible cognitive impairment (i.e. memory loss) will still afflict a substantial proportion of the elderly. While the causes of cognitive impairment in the elderly have yet to be determined with certainty, tools now available favor greater progress in this area of research.

One major challenge in the study of aging is to define the boundaries of normal change as distinct from pathology. An important tool for determining the effects of aging on the brain apart from major neurological diseases is the use of healthy aged laboratory animals, such as rodents and non-human primates. It is reasonable to expect that some features that characterize biological aging of the mammalian brain will be identified using such species as models of the aging human brain. As these animals do not develop neurodegenerative conditions, such as Alzheimer's disease, brain aging can be examined apart from disease. However, aged laboratory animals must be protected against and screened for pathogens as well as physiological conditions and disability, to ensure that studies are uncomplicated by health issues other than aging.

Recent investigations have clearly documented the occurrence of cognitive impairment in aged laboratory animals. For example, behavioral assessments that are sensitive to integrity of the medial temporal lobe system, including the hippocampus, reveal impairments in aged monkeys compared to young adults (Albert and Moss, 1996, Rapp, 1993, Rapp, 1995). Likewise, assessments geared to the same underlying circuitry in rodents reveal age-related impairments (Barnes, 1979, Barnes and McNaughton, 1985, Gallagher et al., 1993). In these cases a common feature is individual variability since every old animal does not perform poorly. Within an aged cohort, deficits are apparent for a substantial subgroup of subjects but some aged animals perform equal to younger subjects (Gallagher et al., 1993). These animal models, which mirror the phenomenon of variability in cognitive decline among elderly humans, indicate that cognitive aging is not inevitable or strictly linked to chronological age. Importantly, such models afford the opportunity to compare the trajectory of changes in the brain that lead to impaired or preserved memory.

Rat models are advantageous in the study of the neurobiology of aging and individual variability because large sample sizes can be used and extensive behavioral characterization can provide a strong background for studies of the brain. Fig. 1(A) shows the individual differences in cognitive decline in aged rats from a behavioral assessment that is sensitive to the function of interconnected structures in the medial temporal lobe, a system that is essential for declarative memory in humans. In this widely used task, referred to as the Morris water maze, rats learn and remember the location of an escape platform guided by a configuration of spatial cues surrounding the maze. Whereas aged rats in the study population shown here have no difficulty swimming to a visible platform, an age-dependent impairment occurs when the platform is camouflaged, requiring the use of spatial information. As shown in the inset in Fig. 1(A), performance for individual aged rats varies greatly, with a proportion of those rats performing on a par with young adults but approximately 40–50% falling outside the range of young performance. The data in Fig. 1(B) demonstrate that this variability among aged rats reflects reliable individual differences. In a reassessment in a new spatial environment several weeks after the original characterization, an aged subgroup is consistently impaired, whereas the aged rats that had preserved behavioral function in the first assessment again perform proficiently. This naturally occurring impairment in an aged population of rodents provides a potential model for examination of the brain changes that underlie age-related cognitive decline.

It was previously thought that neuron loss was an inevitable consequence of aging (Dam, 1979). According to that view, frank neurodegeneration could provide a basis for age-related loss of cognitive function. Recent research, however, has demonstrated that other factors must cause cognitive impairment in behaviorally characterized aged rats. Quantitative unbiased stereology used to determine the number of neurons in components of the medial temporal lobe system revealed that aged rats, irrespective of cognitive status, had comparable cell numbers to those observed in younger animals (Rapp and Gallagher, 1996). This was shown for the principle neurons of the hippocampal formation, including granule cells in the dentate gyrus and pyramidal neurons in the CA fields (Fig. 2) and for neurons in the entorhinal cortex and parahippocampal region; perirhinal and postrhinal cortices (Rapp et al., 2002). These findings were confirmed in other studies of rodents and primates, including humans (Merrill et al., 2001, Rasmussen et al., 1996, West et al., 1993, West et al., 1994). In addition, quantitative Western blot analysis of protein from the hippocampal formation, using antibodies to synaptic markers, revealed no differences in aged versus young rats (Nicolle et al., 1999b, Nicolle et al., 2001a and Fig. 3).

These results indicate that cognitive impairment can occur in aging, independent of neurodegeneration which could involve the loss of neurons or widespread degradation of circuits. These data are consistent with additional findings using this model that within the relevant neural system, no evidence is detected for reactivity to significant injury or degenerative processes by other measures. For example, stereological analysis indicated no change throughout hippocampal formation in number or size of astrocytes (Rapp et al., 1996). Other markers for reactive gliosis, including Ox-44, a marker for microglia, were negative in aged subjects overall including or those aged rats with significant cognitive impairment in this study population (Nicolle et al., 2001c).

These and other findings have led to a shift in focus on the probable basis for age-related cognitive decline. Sufficient data now indicate that critical functional features of neurons in the aged brain are likely to cause cognitive impairment. In particular, the behavioral characterization of individual differences is providing new information about the properties of neurons in the aged brain that differ in cases where cognitive impairment occurs relative to cases where faculties are preserved.

Electrophysiological recordings in animals being behaviorally tested provide an informative view of the encoding properties of neurons and how aging affects the performance of identified neural circuits. Neurons in the rat hippocampus normally encode spatial information characterized by localized activity (place fields) when rats freely explore their environment. Studies in a number of laboratories indicate that place fields are as commonly found in the hippocampus irrespective of age or the cognitive ability of older rats (Barnes et al., 1997, Shen et al., 1997, Tanila et al., 1997a). Notable differences, however, are seen in the dynamic properties of place fields, with evidence that place fields are sometimes less stable in older than in young rats (Barnes et al., 1997), and that place fields also appear more rigid in circumstances where spatial encoding in young rats is rapidly modified (Tanila et al., 1997a, Tanila et al., 1997b, Wilson et al., 2002). This work points to defects in neural mechanisms that ensure both stable representations and flexible modifications in representations. Indeed, further evidence is accumulating to show that specific signaling pathways important for neurotransmission and synaptic plasticity are affected in the hippocampal system (Campbell et al., 1996, Chouinard et al., 1995, Colombo et al., 1997, Landfield and Pitler, 1984, Moore et al., 1993, Nicolle et al., 1999a, Nicolle et al., 1999b, Norris et al., 1996, Shen and Barnes, 1996). Some pathways have been shown to be affected in relation to the severity of behavioral impairment among aged rats (Fig. 4, Chouinard et al., 1995, Colombo et al., 1997, Nicolle et al., 1999a).

Glucocorticoid exposure has been suggested as a key factor contributing to neurodegenerative processes during aging (for recent review McEwen 1999). The effect of glucocorticoids may be due to impaired negative feedback mediated by hippocampal influence on the hypothalamic-pituitary-adrenal (HPA) axis, leading to excessive glucocorticoid production. Evidence for an alteration in targets for glucocorticoids in hippocampal neurons in the aged brain includes a reduction in glucocorticoid receptors (GRs) (Issa et al., 1990, Morano et al., 1994) and more recently that stress induces deficient translocation of GRs and DNA binding to a glucocorticoid response element (GRE) in nuclear extracts of the hippocampal formation (Murphy et al., 2002). Mechanistically, these effects of aging could account for a reduction in the negative feedback on the HPA axis when corticosterone levels are elevated by stress. However, given this evidence for a change in glucocorticoid-mediated mechanisms, it is less clear how targets for glucocorticoids that are associated with hippocampal neurons factor into mechanisms underlying cognitive dysfunction.

Recently, we have begun an evaluation of glucocorticoid-mediated mechanisms in our aged Long-Evans rat model where net loss of hippocampal neurons does not accompany cognitive decline. Our findings demonstrate that neither basal nor peak, acute stress induced levels of circulating glucocorticoid differ in young compared with aged rats either with (aged impaired) or without (aged unimpaired) cognitive deficits, but that aged impaired rats do show a prolonged elevation in circulating glucocorticoid after an acute stress (Bizon et al., 2002). This suggests that functional aging of the hippocampus is associated with perturbed regulation of the HPA axis but that this is not necessarily accompanied by neuronal loss or long term elevations in basal glucocorticoids. Indeed, these results are also consistent with recent findings in mouse models with targeted disruption of genes encoding the high affinity mineralocorticoid receptor (MR) and lower affinity GR. Such findings indicate that although MR may be required for normal neurogenesis or normal numbers of granule cells within denate gyrus (Gass et al., 2000), neuron specific deletion of GR elicited no overt abnormalities in hippocampal morphology (Reichardt et al., 2000). Thus more mechanistic information on glucocorticoid function is needed to precisely define the role of the HPA axis and GR in age associated cognitive decline, apart from frank neurodegenerative mechanisms.

While it has been reported that aged rats show reduced GRE binding in response to stress, we have found substantially higher levels of nuclear GRE binding activity in aged hippocampus than young in a basal non-stressed state (Hoyt et al., 2001). The finding of increased GRE binding activity in the basal state would be potentially consistent with the notion that elevated GR mediated transcription is a factor that contributes to the effects of aging. It is notable, however, that total levels of nuclear GRE binding activity did not differ between aged impaired and unimpaired rats. The somewhat surprising finding of equivalently elevated GRE binding in both impaired and unimpaired aged rats may indicate that cognitive decline is not an inevitable consequence of increased glucocorticoid driven gene transcription in hippocampus. However, more information about relative levels of GR compared with MR in the setting of the aged brain and, more specifically, about transcriptional activities and composition of GR and MR containing transcription complexes at GREs, could provide important information about the functional differences in glucocorticoid action in aged impaired versus unimpaired animals.

Recent studies in our lab have added another level of complexity to possible functional changes in transcription in aging since NFkappaB (NFκB), a transcription factor increasingly implicated in neuroprotection (Mattson et al., 2000), is known to associate with GR containing transcription complexes and to antagonize GR mediated transcription (McKay and Cidlowski, 2000). As with GRE binding activity, aged rats in our study population show increased activity of nuclear proteins which bind to an NFκB response element (NFκB-RE), but again these levels do not differ between aged impaired and aged unimpaired in the hippocampus (Hoyt et al., 2000). In light of these findings, and given the mutual antagonism between GR and NFκB as mediators of transcription, it will be of great interest to establish if aged impaired and unimpaired rats show differences in the degree to which NFκB associates with and functionally influences transcription mediated by GR containing complexes. One approach to this question is to biochemically and quantitatively analyze the components of nuclear GRE or NFκB binding activities in hippocampus of aged impaired and unimpaired rats. Another is to undertake a genomics approach to define patterns of basal, glucocorticoid regulated-, or NFκB regulated-, gene transcription as a direct and more comprehensive measure of transcriptional outcomes that characterize age-induced changes in hippocampus which are associated with impaired or preserved cognitive function.

The use of gene expression arrays offers the potential to simultaneously analyze thousands of genes in order to gain a genetic template of age- and behavior-associated changes in the hippocampal formation. Such an approach also presents some challenges. Firstly, our rat model, like the aging human population comprises a genetically outbred population which can add individual variability as a confounding factor in gene expression profiling. Secondly, using traditional methods to assess levels of specific mRNAs in hippocampus of young, AU and AI hippocampus, we and others have found that age and behavior related changes in gene expression are often relatively small, smaller than the two fold differences in levels of gene expression which have been reported as the limits of discriminating power in existing Genechip or microarray approaches. Recently we employed strategies which appear to overcome such challenges, demonstrating reliable detection of small changes in expression of genes which we have previously shown by traditional methods to differ between aged and young rats (Fig. 5). Selected analyses of a wider range of genes indicated that this method reproducibly reveals a substantial number of genes which show age-related changes in expression within the hippocampus and that are associated with behavioral status. Many of these genes encode proteins which have been functionally linked to age induced impairment in other systems or to regulation of neuronal function and integrity. Other genes have as yet unknown function and no previously established link to aging and the nervous system. These initial studies suggest that the gene expression approach offers great promise into elucidating mechanisms of hippocampal aging and memory loss. The greatest challenge in such approaches lies in optimal data analyses, experimental design and innovative approaches to test the functional significance of altered gene expression profiles.

Section snippets

Conclusion

Research using animal models to investigate aging is informative when all levels of biological and behavioral systems are considered. Such an approach is necessary to understand the proximate basis of altered brain function that disturbs cognitive capacity, a process associated with aging but not strictly tied to chronological age. Such research has the potential for defining the neurobiological basis of the aged impaired phenotype (relative to unimpaired aged phenotype and young) at circuit,

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