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

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.

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 ×40. 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 (Figure 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 (Figure 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 (Figure 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.

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Figure 2

MAP2 optical density values (mean ± SEM) in the brain regions studied. Note the significant reduction in MAP2 immunore-activity in old rats in the hippocampal CA1 sector. ^∗ p < 0.05 vs young rats.

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Table 1

Immunohistochemical standard procedure

Perfusion	Brief flush of 0.9% saline solution followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4
Cryoprotection	30% sucrose in phosphate buffer overnight at 4C
Sectioning	20 μm by a cryostat
Blocking serum	1.5% normal horse serum containing 0.3% Triton X-100 in PBS for 30 min
Primary antibody	Anti-MAP2 monoclonal antibody diluted 1:1000 in blocking serum for 48 hr at 4C (Clone HM-2; Sigma, St Louis, MO)
Biotinylated antibody	1:200 in PBS for 1 hr
Hydrogen peroxide	0.3% in PBS for 30 min
Avidin–biotin complex	1:50 in PBS for 1 hr
DAB	0.05% in PBS activated with 0.01% H2O2 for 10 min
Dehydratation	Ethanol series (70%, 90%, 100%)
Coverslipping	DPX mountant

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.