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. 2007 Aug 21;28(2):277–291. doi: 10.1007/s10571-007-9180-y

Effects of Glucocorticoids on Age-Related Impairments of Hippocampal Structure and Function in Mice

Wen-Bin He 1,2, Jun-Long Zhang 2, Jin-Feng Hu 1, Yun Zhang 1, Takeo Machida 3, Nai-Hong Chen 1,✉
PMCID: PMC11515799  PMID: 17710532

Abstract

Effects of glucocorticoids (GCs) on maze-learning performances and hippocampal morphology were observed in male C57BL/6Cr mice. Correlations between aging, GCs and maze-learning performances were also studied. (2) Eight-arm radial maze was used in maze-learning tests. Learning performance was assessed by the parameters of time of getting all the bait, number of reentry errors into the already-entered arm with bait, and number of missed entries into an unbaited arm. Brain sections, 8 μm thick, were Nissl-stained with cresyl violet or stained immunocytochemically with antibodies against neurofilaments. (3) With aging, normal pyramidal cells decreased gradually in amount, and degenerating cells increased since the age of 18 months, accompanied with the maze-learning deficit. Here we have suggested that these changes were associated with the age-related deficits in adaptation tolerance of neurons to stress. In addition, the age-related deficits in plasticity of hippocampal neurons to GCs in young mice (3 months of age) resulted in an increase in plasma corticosterone (CORT) concentrations, degeneration of hippocampal pyramidal cells, as well as maze-learning deficits. (4) In conclusion, our data indicated that CORT caused the degeneration of hippocampal pyramidal cells and the impairment of memory.

Keywords: GCs, Aging, Hippocampus, Pyramidal cells, Maze learning, C57BL/6Cr mice

Introduction

The increasing population growth in our society has been a question of common interests in the aging process, perhaps most notably the aging of the brain and its crucial cognitive and sensory functions. It has been known that learning and maintenance of spatial memory are controlled by the limbic system of the brain. In particular, the hippocampus, a portion of the limbic system, plays an important role in the function of learning and memory (Manns and Eichenbaum 2006; Shapiro et al. 2006). Clinical reports and experimental data in animal models of aging have shown that age-associated memory deficits are broadly identical to those induced by damage to the hippocampus. Several studies have demonstrated that intrahippocampal injection of colchicine produced both hippocampal and extrahippocampal damages, which are associated with impaired spatial learning both in Morris water and radial arm mazes (Sutherland et al. 1983; Jarrard et al. 1984; McNaughton et al. 1989). The spatial memory and declarative memory could be impaired when the hippocampus was damaged (Bunsey and Eichenbaum 1996). As cognitive function depends on the structural integrity of the hippocampal formation, Kadar et al. (1990) noted that hippocampal morphological changes in rats of different ages corresponded well not only with the cognitive impairments, but also with high correlation especially to lesions at the CA3 region.

Much evidence, from a broad spectrum of studies, has indicated glucocorticoids (GCs) play a key role in aging of the brain, particularly in the hippocampus, a region well documented to show age-related changes in structure and function. It has been proposed that elevated levels of GCs may be neurotoxic, which can endanger neurons by lowering the threshold of damage inflicted by other toxins (e.g., excitatory amino acids), thus influence neuronal structure and its remodeling, interfere with elctrophysiological processes thought as the mechanisms underlying learning and memory (such as long-term potentiation (LTP) and depression (LTD)), and affect neuronal energy balance (Miller and O′Callaghan 2005; Kim et al. 2006). The hippocampus is a well-studied structure in the medial temporal lobe and contains the highest density of receptors for GCs in the brain. It is involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and in aspects of learning and memory. It therefore, acts as a site, where stress hormones feed back upon their physiological regulatory pathway and on behavior (de Kloet et al. 2007).

Excessive exposure to GCs exerts deleterious effects on the hippocampus of the brain. Long-term exposure to elevated corticosterone levels resulted in spatial learning deficits in the rats of different age (Sapolsky et al. 1985; Yau et al. 1995). However, attenuating corticosterone levels can prevent high corticosterone-induced impairments in spatial memory (Wright et al. 2006). In human beings, it has been noticed that some diseases associated with memory are related to hippocampus. Compared with age-matched people, the number of hippocampal neurons in the brain of the Alzheimer patients decreased significantly and the pathological changes of neurofibrills were clearly visible in hippocampus (Noda et al. 2006). The age-related deficits of learning and memory functions seem related to the limbic system of the brain, particularly to hippocampus. The decline of age-related learning and memory may be correlative with the decrease in the number of receptor and the death of neurons induced by GCs (Murialdo et al. 2000; Elgh et al. 2006).

This study was, therefore, dedicated to observe the effects of GCs upon maze-learning performances and morphology of hippocampal neurons in mice. Our main purpose is to clarify the relation between maze learning, aging, and GCs.

Methods

Animals

This study was approved by the Animal research committee of Institute of Materia Medica, Chinese Academy of Medical Sciences. All C57BL/6CrSlc mice used in this study were purchased from Institute of Experimental Animal, Chinese Academy of Medical Sciences, and bred in our laboratory. Animals were housed three in one polycarbonate cage with length of 22 cm, width of 15 cm and height of 12 cm. They were maintained on a 12:12 light/dark schedule (lights on at 08:00) with 22 ± 1°C temperature and 50% humidity. Bait and water were covered. Only in the 8-arm radial maze task period, but not during other experimental periods the animals were placed under restricted food intake. All animals had free access to water throughout the experimental periods.

Experimental Design and Treatment

According to our design, this study includes four parts: (1) age-related changes of hippocampal structure and maze learning performances in mice; (2) effects of acute GCs adminisitration on the morphology of hippocampal pyramidal cells in mice; (3) effects of short-term GCs administration on hippocampal structure and maze learning performances in mice and (4) effects of long-term GCs administration on hippocampal structure and maze learning performances in mice.

For part (1), Animals were divided into the age groups of 3, 12, 18, 24, and 30 months. Then, 8-arm radial maze task was performed followed by histology detected.

In the experiment of part (2), male mice of 80 days of age were used. Mice were divided into two groups: oil group (n = 12) and CORT group (n = 18). Mice in CORT group were injected subcutaneously with 6.7 mg CORT/ml olive oil at 18:00. In oil group 1 ml of olive oil was injected in mice. About 1 h after injection and in the 2 h, 1st, 2nd, 3rd, 6th, 10th, and 14th day, blood samples were collected in 18:00–20:00. The samples were rapidly separated by centrifugation at 15,000 rpm for 5 min at 4°C, and 0.4–0.5 ml of serum was stored as sample at −15°C until assay. Corticosterone levels were measured by radioimmunoassay (RIA). Brain sections were examined and the pyramidal cells in CA1, CA2, CA3, and CA4 were counted from 10 sections per each brain.

Male mice of 80 days were also used in the experiment of part (3). Animals were divided into three groups. CORT group: animals were injected subcutaneously with 6.7 mg CORT/ml olive oil (n = 8); vehicle group: animals were injected with 1 ml of olive oil (n = 8); intact group: unhandled, unstressed mice (n = 8). From the third day after injection, maze test was performed then brain sections were observed.

In part (4), subjects were 50-day-old male mice. Animals were injected subcutaneously with 1 mg CORT/0.2 ml olive oil every other day (n = 8); controls were injected only with 0.2 ml of oil. Maze learning test began from 90th day followed by histology assay.

Performance of Eight Radial Maze Task

The maze that was elevated 50 cm off the ground has eight radial arms (30 cm long and 5 cm wide) from a central platform. There were holes at the end of each arm, in which a piece of chow was set. In our test, chow was placed in holes of the 1st, 3rd, 5th, 6th, 7th arms, holes of the 2nd, 4th and 8th arms being with no chow. Animals were trained for 4 days on the maze, and then, maze test was performed for 15 consecutive days in the afternoon. Learning performances were assessed by the following parameters: (1) time for attaining all chows in a session; (2) number of reentry errors into the same arm with chow; (3) number of miss entries into an unbaited arm. The reference marks were kept unchanged during the training and testing period. When a session was completed, the mouse was returned to the home cage and the maze was wiped clean with ethanol before the next test.

Histological and Immunocytochemical Assay

After the completion of the testing program, animals were fixed by perfusion with 10% formalin. The excised brains were put in 10% formalin to postfix for 2 weeks, then dehydrated in the gradient alcohol (from 35% to 100%) and benzene, and embedded in paraffin. Sections were cut at 8 μm in cross section and Nissl-stained with cresyl violet. In order to discriminate the pyramidal neurons from the glial cells, some sections were stained immunocytochemically. Standard immunocytochemical procedures were used herein. Briefly, sections were washed in phosphate buffered saline (PBS, pH 7.4) containing 0.75% hydrogen peroxide and 0.1% ammonium citrate for 30 min. Then the sections were blocked in PBS containing 10% normal goat serum for 40 min at room temperature. The sections were incubated overnight at 4°C in the neurofilament triplet protein 200 KDa antibody (1:500, Sigma). The sections were washed in Tris-buffered saline (TBS: 50 mM, pH 7.4) and incubated with secondary antibody, biotinylated goat anti-rabbit IgG (1:250, Vector Laboratories), in TBS and 1% normal goat serum for 60 min at room temperature. After this time, the sections were washed in TBS and incubated in avidin-biotin-peroxidase complex (Vector Laboratories, Standard ABC Peroxidase Kit) for 60 min, followed by washes in TBS and Tris–HCl buffer (50 mM, pH 7.4). Washes throughout the procedure were performed in triplicate. The signal was visualised by incubating sections in 0.05% w/v 3,3V-diaminobenzidine, 0.07% w/v nickel chloride and 0.0021% w/v H2O2 in Tris–HCl. The sections were mounted onto gelatin-coated slides, dehydrated, demyelinated in xylene and coverslipped with Eukitt mountant (BDH, UK). Sections from all experimental conditions were reacted simultaneously to reduce processing variability. The hippocampal pyramidal cells in CA1, CA2, CA3 and CA4 regions and the dentate granule cells were observed.

Cell Count and Observation

Hippocampal middle cross sections were divided for convenience into several subfields, Cornu Ammonis (CA)1 (with small pyramidal cells), CA2 (with two or three layers of large pyramidal cells), CA3 (with four or five layers of large pyramidal cells), CA4 (with a large number of scattered pyramidal cells) (Fig. 1A, B). There are two limbs in dentate gyrus called suprapyramidal blade and infrapyramidal blade (Fig. 1B). Neural projection pathways around the hippocampus are as follows (Fig. 1C). The main afferent of hippocampus reaches dentate gyrus via the perforation fascicles coming from entorhinalis area. Mossy fibers reach the synapses of the pyramidal cells in CA3 connects with the synapse of the pyramidal cells in CA1. Then the axon of pyramidal cells in CA1 conveys massages to the fornix through hipocampal fimbria. In this study, according to the method described by Williams and Rakic (1988) and West and Gundersen (1990), we observed the pyramidal cells and dentate granule cells in the cross sections of the middle portion of a hippocampus that had been sliced into 60 cross sections, 8 μm thick per section. From 25th to 40th sections, five sections were counted in each brain. In order to avoid recounting of the same neuron twice, only the neuron with a clear nucleolus was observed.

Fig. 1.

Fig. 1

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Diagram of hippocampus (A) Corss-section in the middle portion of a hippocampus. (B) Subregions of a hippocampus. CA, Pyramidal layer of Cornu Ammonis (CA); DG, Dentate Gyrus; Sup, Suprapyramidal blade; Inf, Infrapyramidal blade. (C) Major intrinsic connections of the mouse hippocampal formation. (AE, Area Entorhinalis; Per, Perforating Fiber; DG, Dentate Gyrus; MF, Mossy Fibers; CA, Cornu Ammonis; Sch, Schaffer Collaterals; Sub, Subiculun)

Radioimmunoassay for CORT Level

Corticosterone level was measured by RIA kit (Amersham, Biosciences, Japan). In brief, blood sample were collected into tubes containing EDTA (1:200). Samples were centrifuged in 15,000 rpm at 4°C for 5 min. Plasma sample were removed and stored below −15°C until analysis. Label polypropylene or polystyrene tubes in duplicate for total counts (TC), non-specific binding (NSB), zero standard (B0), standards and samples. Pipette 100 μl, 50 μl assay buffer into the NSB tubes and B0 tubes, respectively. Pipette 50 μl of each standard into the appropriately labeled tubes. Pipette 50 μl antiserums into all tubes except NSB and TC. Pipette 50 μl of [125I] CORT into all tubes. The TC tubes should be stopped and put aside for counting. Vortex mix all tubes thoroughly. Cover the tubes and incubate for 3 h at room temperature. Gently shake and swirl the bottle containing Amerlex-M second antibody reagent to ensure a homogeneous suspension followed by incubation at room temperature for 30 min. Separate the antibody bound fraction using either magnetic separation. Centrifuge all tubes in 3,000 rpm at 4°C for 30 min; Any adhering liquid was carefully removed; The radioactivity present in each tube was determined by counting for at least 60 s in a gamma scintillation counter.

Statistical Analysis

Data are expressed as mean ± SD, and comparisons between groups were made using analysis of variance (ANOVA) followed by student’s t-test. Significance was accepted at the P < 0.05 level.

Results

Age-Related Changes of Hippocampal Structure and Maze Learning Performances

There were no differences between the 3- and 12-month-old groups in maze learning performances. Beginning from the 12th month, however, there was a decrease tendency in learning performance, i.e., time for completing a session was prolonged and the number of errors and misses were increased. There was a significant increase in time for completing a session from the age of 18 months, while significant increase in the numbers of errors and misses appears from the age of 24 months (P < 0.05, P < 0.01) (Fig. 2J).

Fig. 2.

Fig. 2

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Age-related changes of hippocampal structure and maze learning performancesA and B indicated CA2 and CA3 region of 3-month-old mice, respectively. C and D showed CA2 and CA3 region of 24-month-old mice, respectively. E and F represented CA2 and CA3 region of 30-month-old mice, respectively. G showed hippocampus and dentate gyrus of 30-month-old mice. H indicated dentate gyrus of 30-month-old mice. Dark triangles indicate dark cells. White triangles showed normal granule cells. Degenerate pyramidal cells showed increased tendency from 3-month-old to 30-month-old in both CA2 and CA3 regions of C57BL/6CrSlc mice. However, neither the number of total cells nor the neuron density is decreased significantly in aging process. Scale bar: 15 μm in A–H; 60 μm in G. (I) Comparison of the number of cells in CA2 and CA3 region in each age group. (J) Comparison of maze learning performances in mice of each age groups. *P < 0.05 **P < 0.01 versus 3-month-old group. *P < 0.05 **P < 0.01 24, 30 versus 3-month-old group

Few dark cells were visible in both layers of pyramidal cells and granule cells in groups of 3-, 12- and 18-month-old. In 24-month-old group, however, massive dark cells were found in CA2 and CA3 regions while dense rows of cells were still visible. No obvious cell loss has been found (Fig. 2A–D). Sections from 30-month-old mice were with more degenerating cells and less normal cells (Fig. 2E–H). In a few sections of 30-month-old group, layers of pyramidal and granule cells were narrowed, rows of cells were scattered and gaps among cells were widened (data not shown). Nevertheless these phenomena were not found in the other sections in the same age group. Cell counts demonstrated that, compared with 3-month-old group, the number of degenerating cells was significantly increased in both groups of 24- and 30-month-old, while the number of normal cells was significantly decreased. No significant decrease was found in the number of total pyramidal cells (Fig. 2I). Immunocytochemically-stained sections indicated that neuron densities were not decreased in 30-month-old group as compared with 3-month-old group (Fig. 3).

Fig. 3.

Fig. 3

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Immunocytochemically-stained sections of CA2 region of 3-month-old (A, C) and 30-month-old (B, D) mice. Massive deep-stained pyramidal cells have been found in 30-month-old group. There are more glia cells in 30-month-old group than that of 3-month-old group. Dark arrows indicate degenerate pyramidal cells; dark triangles indicate glis cells. Scale bars: (A, B) 15 μm; (C, D) 10 μm

Effects of GCs on Hippocampal Pyramidal Cells

After CORT administration but not oil treatment, there were a great number of dark pyramidal cells in each section of CA1, CA2, CA3 and CA4 regions from 2 to 14 days, especially in third day. Moreover, dark pyramidal cells after CORT treatment were especially visible in CA2 and CA3 regions (Fig. 4A, B). These dark pyramidal cells were obviously different from normal pyramidal cells (Fig. 4C). Nuclei of dark pyramidal cells were shrinked and dark-stained, and nucleoli were hard to be identified. Normal pyramidal cells were with round or ellipsoid nuclei, and transparent cytoplasma. Nuclei and nucleoli were clearly visible in normal cells.

Fig. 4.

Fig. 4

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Effects of GCs on hippocampal pyramidal cellsA and B indicated CA2 and CA3 region of hippocampus in 3 days after oil or CORT treatment, respectively. Dark cells in CA2 and CA3 regions are significantly increased after CORT treatment. C indicated the morphology of pyramidal cells. Nucleus of normal pyramidal cells (with white arrow) was clearly visible, while in dark pyramidal cells (with dark arrow), cytoplasm was stained dark and nucleus was hard to be identified. Scale bars: 40 μm in A, B; 10 μm in C. D showed the percentages of dark clles in total number of cells of each regions of hippocampus. *P < 0.05 **P < 0.01 CORT group versus OIL group. E represented the changes of the number of total pyramidal cells in CA1, CA2, CA3 and CA4 regions in 14 days after CORT administration. F showed the changes of plasma CORT level after CORT administration. *P < 0.05 **P < 0.01 CORT versus oil group

Our data showed the percentages of degenerative cells in CA1, CA2, CA3 and CA4 regions in CORT group were significantly higher in 3–7 days after CORT treatment than that in oil group (Fig. 4D). Compared with oil group, from 2 to 14 days after CORT treatment, the percentages of degenerative cells in CA2 and CA3 regions in the CORT group were obviously elevated (Fig. 4D). Nevertheless, total cell number remained unchanged till the 14th day in all of CA1, CA2, CA3 and CA4 regions. Total number of cells was not changed following the decrease in the number of degenerative cells (Fig. 4E).

As shown in Fig. 4F, CORT level was obviously elevated (as high as 29.8 μg/dl). Although the level was decreased at 24 h after injection, it was still significantly higher than that of oil control. This high level was maintained for 2–3 days (Fig. 4F).

Short-Term Effects of GCs on Hippocampal Pyramidal Cells and Maze Learning Performances

Compared with the other groups, time for completing 15 sessions was prolonged in CORT group (P < 0.01) and both number of missing entry (P < 0.05) and number of reentry into the same arm (P < 0.01) were obviously increased. No significant differences were found between intact and vehicle group (Fig. 5E). There was a tendency that, with the increase in the number of sessions, the number of selected arms was decreased gradually in intact and vehicle group but not in CORT group (Fig. 5F).

Fig. 5.

Fig. 5

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Short-term effects of GCs on hippocampal pyramidal cells and maze learning performances. A showed the representative section of CA2 region from oil group in the 22nd day after oil treatment. B and C indicated the representative sections of CA2 region from CORT group in the 3rd day and the 22nd day after CORT administration, respectively. Dark arrows indicate degenerate pyramidal cells; White arrows indicate normal pyramidal cells. Scale bar, 10 μm. D showed the comparison of number of cells in CA2 and CA3 regions. **P < 0.01 CORT group versus OIL group, E indicated the comparison of maze learning performances in various groups. *P < 0.05 **P < 0.01 CORT group versus OIL group, F showed the changes of behavioral performances in 15 consecutive sessions

After maze learning program, hippocampal sections were examined. Nearly no degenerative pyramidal cells were found in the sections of any region in both intact and vehicle groups. In CORT group, dark pyramidal cells were visible in any CA region. These changes were most distinct in CA2 and CA3 regions. Pyramidal cells in CA2 region in intact group were with a transparent cytoplasm, full-sized nucleus and a clear nucleolus, while in CORT group, dark pyramidal cells in CA2 and CA3 regions were with dark-stained cytoplasm, and shrinked nuclei. Moreover, cytoplasm of pyramidal cells in 22 days after CORT treatment was lighter than that in 3 days after CORT treatment (Fig. 5A–C).

In 22 days after CORT treatment, total number of cells remained unchanged in CA1, CA2, CA3 and CA4 regions. On the other hand, the number of dark cells in CA2 and CA3 regions was increased significantly in CORT group (P < 0.01). There were no significant differences in the number of dark cells between intact and vehicle groups (Fig. 5D).

Long-Term Effect of GCs on Hippocampal Pyramidal Cells and Maze Learning Performances

Compared with vehicle group, time for completing 15 consecutive sessions was prolonged in CORT group (P < 0.001). Both the number of misses and the number of errors were increased (P < 0.01, P < 0.05) (Fig. 6H).

Fig. 6.

Fig. 6

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Long-term effects of GCs on hippocampal pyramidal cells and maze learning performancesA and C showed the representative sections of hippocampal CA2 and CA3 regions in Oil group, respectively. B and D indicated the representative sections of hippocamal CA2 and CA3 regions in CORT group. Dark triangles in B and D indicate dark pyramidal cells. E showed the morphology of pyramidal cells. Dark arrow in E indicated dark pyramidal cell and white arrow indicated normal pyramidal cell. F showed the representative morphology of hippocampus and dentate gyrus after long-term CORT treatment. Dark triangles in F indicated that dark cells could be found in each hippocampal CA1, CA2, and CA3 regions and dentate gyrus. Scale bars: 20 μm in A–D; 10 μm in E; 20 μm in F. G showed the comparison of the number of cells between CORT and OIL group. P < 0.01 CORT group versus OIL group. H indicated the comparison of maze learning performances in OIL and CORT groups. *P < 0.05, **P < 0.01, ***P < 0.001 CORT versus OIL group

In vehicle group, only a few dark pyramidal cells were found in hippocampal CA2 and CA3 regions (Fig. 6A, C). On the other hand, numerous dark cells were found in CA2 and CA3 regions of CORT group (Fig. 6B, D). As shown in Fig. 6E, we found that dark pyramidal cells in CA2 and CA3 regions of CORT group were shrinked, gaps among cells were widened, cytoplasm was dark-stained, and nucleoli and chromonema were difficult to be discriminated. However, normal pyramidal cells in CA2 and CA3 regions were swollen and full-sized, linked up clearly, and with nucleoli and chromonema visible. In dentate granule cell layer and CA1 regions, numerous dark cells were observed in CORT group (Fig. 6F). Moreover, even in the immunocytochemically-stained sections, no cell loss or decrease in neuronal populations was found (Fig. 7).

Fig. 7.

Fig. 7

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Immunocytochemically-stained sections of hippocampus in long-term CORT administrated mice. A showed the hippocampal CA1, CA2, CA3 and CA4 regions and the dentate gyrus in OIL group. B represented the hippocampal CA2 and CA3 regions in CORT group. Dark arrows indicated degenerate pyramidal cells. C and D indicated the representative morphology of CA2 regions in OIL group and CORT group, respectively. Dark arrows indicated degenerate pyramidal cells and white arrows indicated glia cell. Scale bars: (A) 150 μm; (B) 25 μm; (C, D), 10 μm

Cell count data shown the number of dark cells in CA2 and CA3 regions of CORT group increased significantly more than that of vehicle group (P < 0.01). However, total number of pyramidal cells was not decreased. Even in dentate gyrus of CORT group, the number of dark granule cells was increased, although no significant increase was found in total number of pyramidal cells between CORT group and vehicle group (Fig. 6G).

Discussion

GCs secreted by the adrenal cortex are important regulators of homeostasis. It has been demonstrated GCs protect adult animals from acute catabolic consequences of stress (Tasker 2006). CORT concentration could be elevated with aging in rats (Landfield et al. 1978). GCs also have the potential to cause hippocampal damage. High level of these hormones was found to generate aging-like morphological changes in the brain (Landfield et al. 1978; Hibberd et al. 2000; Montaron et al. 2006). We observed that CORT level of older mice was a little higher than that of younger ones.

The hippocampus is a brain region involved in learning and memory. It also plays a key role in the endocrine functions of the hypothalamic-pituitary-adrenal axis (Miller and O′Callaghan 2005; Magri et al. 2006). As it is a principal neural target tissue for GCs, it is, therefore, reasonable to assume that such action may lead to specific cognitive deficits. Accumulated study showed that there was a loss in hippocampal pyramidal cells and decline in learning performance with aging (Landfield et al. 1981a, b; Sapolsky et al. 1985; Shankar et al. 1998; Miki et al. 2004; von Bohlen et al. 2006). Our observations demonstrated that, compared with young mice, total number of cells was not decrease in aged mice, although there was increase in dark cells and decrease in normal cells. Several aged mice (30 months old) showed a loss of pyramidal cells, but it was not significant, when compared with young mice. Correlation between the changes in behavior and structure in the aged brain has been studied. It was found that the decrease in the number of perforated synapse of the dentate gyrus was correlated with the degree of memory impairment in aged rats (Geinisman et al. 1986, 2004; Nicholson et al. 2004). Kadar et al. (1990) showed that significant memory impairments began at the age of 12 months in Wistar rats, and further impairments occurred at the age of 24 months. It has been known that maze learning performance was associated with aging. In our study, although time for completing a session was prolonged at 18 months of age, the other two parameters remained unchanged. It seems that maze learning performances were not declined at the age of 18 months in mice. Actually, all the three parameters (time, number of errors and misses) obviously changed in the age of 24 months. Moreover, brain sections showed a massive increase in dark cells and apparent decrease in normal cells. It indicated that significant memory deficits began from the age of 24 months. Our data also showed there was a parallel correlation between hippocampal morphological changes and maze learning performances, which was consistent with previous reports concerned (Geinisman et al. 1986; Kadar et al. 1990). The changes in hippocampal CA regions induced by short-term CORT administration, i.e. an increase in dark cells in CA2 and CA3 regions, were consistent with the earlier findings that hippocampal cells are subject to short-term effects of physiological level of CORT (Miller et al. 1989; Takahashi 1995). We have observed that there are some interactions between dark cells and high level of CORT, which was induced by acute excess injection. However, it still needs to be further elucidated. Otherwise, neither granule cell nor pyramidal cell loss was found in our study. Nissl staining was used in our study to discriminate if the neuron degeneration would occur. When neurons appear degenerative changes, it will show dark stain. However, the normal neurons will show light stain. As the number of dark cells was apparently increased after CORT administration, it is possible that cognitive memory can be affected by degenerated cells.

Woolley et al. reported that chronic administration of high level of corticosterone to the adult rat resulted in an apparent atrophy of apical dendrite tree in CA3 pyramidal cells (Woolley et al. 1990) Moreover, elevated GCs levels were implicated in hippocampal neurons loss (Landfield et al. 1981a, b; Sapolsky and Pulsinelli 1985). Long-term exposure to elevated GCs level resulted in spatial learning deficits in mid-aged rats (Bodnoff et al. 1995). Our data, coincident with Bodnoff et al. (1995), suggested that memory impairments did not appear to be the consequence of hippocampal neuron loss, which indicated that memory impairment resulted from chronic GCs treatment may not be accompanied with loss of hippocampal neurons. Although the mechanisms of neuronal degeneration induced by CORT treatment are not very clear, it has been suggested that these changes are mainly visible in hippocampal pyramidal cells, differing from the degeneration induced by transient ischemia, which may also be found in glial cells (Deshpande et al. 1992; Fukuda et al. 1993).

In summary, our observations on C57BL/6Cr mice indicated: (1) with aging, the number of dark cells increased and maze-learning performances decreased. As both changes were obviously observed in the age of 24 months, there were some correlation between hippocampal morphology and learning and memory; (2) the degeneration of hippocampal neurons can be accelerated by long-term or short-term GCs administration; (3) massive degeneration of pyramidal and granule cells may result in memory impairment, suggesting that both hippocampus and dentate gyrus were involved in the process of learning and memory.

Acknowledgements

This work was supported by the National Basic Research Program (973 program, Grant No. 2004CB518906), Hi-Tech Research and Development Program of China (863 program, Grant No. 2006AA020501), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, Grant No. IRT0514), National Natural Science Foundation of China (NSFC, Grant No. 30572342), and Natural Science Foundation of Shanxi Province, China (Grant No. 2006011101). We have also expressed gratitude to Dr. Chia Fuhzah Njiti, working at International Medical Educational Center, Shanxi College of Traditional Chinese Medicine, for his heartful revising of the manuscript.

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