Enriched environmental conditions reverse age-dependent gliosis and losses of neurofilaments and extracellular matrix components but do not alter lipofuscin accumulation in the hindlimb area of the aging rat brain

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Abstract

We provide a description of a correlation of lipofuscin accumulation and expression of glial fibrillary acidic protein in the cerebral cortex of aged rats. Glial fibrillary acidic protein showed a complementary distribution pattern to perineuronal nets, visualized with Wisteria floribunda agglutinin. With progressing age (12–36 months), a strong increase of lipofuscin and gliosis occurred in functionally characterized cortical areas, whereas a concomitant, area-specific loss of perineuronal nets was found in the cortical somatosensory representation of the hindlimbs. In contrast to lipofuscin accumulation and increased gliosis, the loss of perineuronal nets and the reduction of non-phosphorylated neurofilament H were in part reduced or prevented by housing the animals under enriched environmental conditions between 33 and 36 months of age. Especially the reduction of astrocytosis by 20% which coincided with a reduction in the loss of extracellular matrix components involved in forming the glia–neuron-interface demonstrates, that the aging cortex retains its potential for functional plasticity.

Introduction

On a behavioral level, old rats are known to show a number of age-related changes (O'Hare et al., 1999, Pelosi and Blumhardt, 1999), such as a characteristic impairment of the sensorimotor system, which is most strikingly expressed in a walking impairment involving the hindlimbs (Schuurman et al., 1987, Ingram, 1988, Stoll et al., 1996). Our studies (Spengler et al., 1995) also revealed profound age-related electrophysiological alterations of the cortical hindpaw representation of aged rats reflected by a severalfold enlargement of their receptive field representations. When such old rats became transferred to an enriched environment, receptive fields of neurons in the cortical hindpaw representation area decreased again to nearly the size found in younger adults. Therefore, the question arises as to whether these electrophysiological and functional findings would be accompanied by intracortical, morphological alterations. In addition, it is important to clarify whether these changes are caused by more general, age-related degenerative processes, or whether they truly are use-dependent alterations.

We postulate, that some aspects of age-related changes reflect plastic reorganization as a consequence of prolonged disuse of the hindlimbs rather than a process of degeneration. This hypothesis is based on the fact that in 3-year-old rats the sensorimotor behavior of the forepaw remains largely unaffected. If solely degeneration were responsible for age-related alterations, one would expect similar changes to occur in the fore- and the hindpaw representation areas. Electrophysiological analysis of the area of forepaw representation in a previous study using exactly the same experimental setup and rat strain, revealed, however, no age-related alterations of the receptive fields or the respective cortical representational maps (Berkefeld et al., 1996). We have expanded this model by introducing anatomical techniques to investigate the possible cytoarchitectonic and anatomical basis for the electrophysiologically observed functional changes.

Nerve cells within the cortex of man are known to accumulate lipofuscin during their life span. The amounts and the characteristic patterns of that pigment distribution may serve as criteria for staging of age-induced alterations in the human brain (Braak, 1979, Braak, 1983). Only very few animal models for pigment accumulation in the brain are described (Braak et al., 1984, Koppang, 1973/4, Purpura and Baker, 1978). Besides the pattern of lipofuscin accumulation, the distribution of reactive glia and the glial–neuron-interface (Celio et al., 1998) were analyzed. Glial fibrillary acidic protein (GFAP) has been shown to be expressed by reactive astrocytes (Bignami and Dahl, 1976, Eng and Dearmond, 1983, Eng et al., 1987, Eng, 1988). GFAP positive astrocytes are responsible for an age-related, chronically developing gliosis (Murphy, 1993). In normal tissue, glial cells are involved in establishing a more specialized glial–neuron-interface in the direct environment of synaptic contacts. This glial–neuron-interface which is part of the extracellular matrix (Celio et al., 1998), can be visualized by means of lectin binding (Naegele and Katz, 1990, Härtig et al., 1992, Härtig et al., 1999, Brückner et al., 1994, Seeger et al., 1996). Similar to previous reports, Wisteria floribunda-agglutinin (WFA) was used to study age-related alterations of this matrix component. Among others, the interaction between glia and neuron by means of extracellular matrix is important for the maintenance of the acid–base steady-state and is subject to modification by pH and microenvironmental changes (Chesler, 1990, Härtig et al., 1999). In addition, and in order to also focus on neurons, neurofilamental alterations were studied. Neurofilaments and their state of phosphorylation have been identified as sensitive markers of pathology related as well as during regeneration processes (Wong et al., 1995). Furthermore, transgenic mouse models in which the heavy or light subunits of neurofilament protein are overexpressed, display a motor neuron disease similar to amyotrophic lateral sclerosis. Therefore, cytoskeletal alterations (both hypo- and hyperphosphorylations) were described as being a defining attribute of the degenerative processes (Morrison and Hof, 1997). In Alzheimer's disease neurofibrillary tangles occur primarily in neurofilament-protein-rich neurons by aggregation of cytoskeletal elements into tangles, indicating that alterations within neurofilaments may be good candidates to detect age-related changes at the neuronal level.

Section snippets

Experimental animals

We used 45 hybrid Fischer 344* Brown Norway rats (FBNF1 rats) as described by Van der Staay and Blokland (1996) of following ages: 3 months (three animals as controls), 11–12 months (nine animals), 24 months (nine animals), 36 months (16 animals divided into eight animals housed in standard environment and and eight animals housed under enriched living conditions during the last 3 months of their life). Groups of 4–5 control rats were kept under standard conditions in littered standard Type IV

Patterns of lipofuscin accumulation and GFAP expression

An age-dependent accumulation of lipofuscin and GFAP expression were one of the most evident signs of aging. The first deposits of dusty lipofuscin granules in the rat brain occurred at the age of about 1 year in most cortical regions. Small amounts of lipofuscin were co-distributed with a few GFAP expressing astrocytes. The pattern of lipofuscin distribution was more homogeneous than has been described for the aged human brain (Braak, 1983), and did not allow the establishment of a

Discussion

Over the last two decades, the availability of rodent models has greatly aided research progress in the neurobiology of aging (Ingram and Jucker, 1999). Rats have been the primary models of choice. In general, their behavior and their brain organization can differ decisively from that of humans. However, beyond cognitive aspects, locomotory activity in rats as a marker of vigilance and level of sensorimotor performance provides an interesting feature, that can be studied during postnatal

Acknowledgements

We thank Dr N. Palomero-Gallagher for helpful suggestions and corrections. The study was supported by DFG 314/10 (H.D.) and SFB 194-B2 (H.-J. B.).

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