Journal of Histochemistry and Cytochemistry, Vol. 49, 1065-1066, August 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Distribution of MAP2 in Hippocampus and Cerebellum of Young and Old Rats by Quantitative Immunohistochemistry

Giuseppina Di Stefanoa, Tiziana Casolia, Patrizia Fattorettia, Natascia Gracciottia, Moreno Solazzia, and Carlo Bertoni–Freddaria
a Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, 60121, Ancona, Italy

Correspondence to: Giuseppina Di Stefano, Neurobiology of Aging Lab., INRCA Research Department, Via Birarelli 8, Ancona AN 60121, Italy. E-mail: g.distefano@inrca.it


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The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging. (J Histochem Cytochem 49:1065–1066, 2001)

Key Words: microtubules, MAP2, hippocampus, cerebellum


  Introduction
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Summary
Introduction
Literature Cited

THE microtubule-associated protein MAP2 is a cytoskeletal protein localized in the neuronal dendritic compartment. It is considered a marker of structural integrity because it is involved in morphological stabilization of dendritic processes. Moreover, MAP2 expression coincides with dendritic outgrowth, branching, and post-lesion dendritic remodeling, suggesting that this protein plays a crucial role in plasticity (Johnson and Jope 1992 ). A reduction in MAP2 content has been reported in microtubules isolated from whole aged rat brain, and it has been correlated to impaired microtubule assembly observed in old age (Matus and Green 1987 ). Loss of MAP2 immunoreactivity in the hippocampus and parietal cortex of old rats has been observed in a recent immunohistochemical study (Chauhan and Siegel 1997 ). To evaluate whether MAP2 can be considered an index of reduced dendritic integrity and plasticity in aging, we performed a quantitative immunohistochemical study of MAP2 distribution in hippocampus and cerebellum of young and old rats.

Two groups of female Wistar rats (3 and 28 months of age; n=4 each) were selected and sacrificed according to the guidelines of Italian Ministry of Health regarding the use of laboratory animals. They were anesthetized with 200 mg/kg body weight of 2,2,2 tribromoethanol and perfused intracardially. Hippocampus and cerebellum were removed and processed for the immunohistochemical reaction (Table 1). Incubation in mouse IgG1 replaced anti-MAP2 antibody in control samples.


 
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Table 1. Immunohistochemical standard procedure

Quantitative evaluation of the immunohistochemical reaction was performed by optical density (OD) measurements using a Kontron Imaging System KS300 connected to a Leitz Laborlux light microscope. The light microscope condenser was set at minimum brightness, the voltage was fixed at 4.5 V, and the objective lens used was x40. Five measurements were made on each section and three sections were analyzed per animal. Measurements were performed in cerebellar cortex molecular layer and hippocampal CA1 sector. Optical densities of control sections were subtracted and the results were expressed as mean ± SEM. Data were analyzed with one-way ANOVA followed by the Student–Newman–Keuls test. p values of <0.05 were considered statistically significant.

All regions examined showed a pattern of intense MAP2 staining in dendrites and a weaker staining in cell bodies. The dendrites of CA1 pyramidal neurons were more intensly stained than granule cell dendrites (Fig 1A). In the molecular layer of cerebellar cortex, MAP2 immunoreactivity was characterized by intense labeling of distal Purkinje cell dendrites, whereas Purkinje cell bodies and proximal dendrites were faintly stained (Fig 1B). Densitometric analysis showed a significant reduction in MAP2 immunoreactivity in hippocampal CA1 region in old rats compared to young ones. No age-dependent change was observed in cerebellar MAP2 distribution (Fig 2).



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Figure 1. Distribution of MAP2 immunostaining. (A) Hippocampal coronal section. Dendrites of pyramidal cell neurons in CA1 field (sr, stratum radiatum) are very intensely stained compared to granule cell dendrites (ml, molecular layer). (B) Cerebellar cortex stained with anti-MAP2 showing the weak staining of Purkinje cell bodies (P) and proximal dendrites and the marked labeling of MAP2 in the outer molecular layer (oml). Bars: A = 150 µm; B = 40 µm.

Figure 2. MAP2 optical density values (mean ± SEM) in the brain regions studied. Note the significant reduction in MAP2 immunoreactivity in old rats in the hippocampal CA1 sector. *p<0.05 vs young rats.

Our preliminary data show that MAP2 expression decreases with advancing age in hippocampus but not in cerebellum. According to the role of MAP2 in dendritic stability and plasticity, our results suggest that selected areas of the rat brain are impaired, whereas others maintain their structural integrity and the ability to sustain dendritic remodeling in aging. The immunostaining density for MAP2 in hippocampal CA1 field is reduced in old rats compared to young ones. These data suggest that the decrease of MAP2 may be associated with age-related impairment of memory functions. Taken together, our findings indicate that MAP2 can be considered a marker for the age-dependent deterioration of dendritic stability and plasticity.

Received for publication November 28, 2000; accepted February 16, 2001.
  Literature Cited
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Summary
Introduction
Literature Cited

Chauhan N, Siegel G (1997) Age-dependent organotypic expression of microtubules-associated proteins (MAP1, MAP2 and MAP5) in rat brain. Neurochem Res 22:713-719[Medline]

Johnson GVW, Jope RS (1992) The role of MAP2 in neuronal growth, plasticity and degeneration. J Neurosci Res 33:505-512[Medline]

Matus A, Green GDJ (1987) Age-related increase in a cathepsin D like protease that degrades brain microtubules-associated proteins. Biochemistry 26:8083-8086[Medline]