Abstract

In aging individuals, age-related cognitive decline is the most common cause of memory impairment. Among the remedies, ginsenoside Rg1, a major active component of ginseng, is often recommended for its antiaging effects. However, its role in improving cognitive decline during normal aging remains unknown and its molecular mechanism partially understood. This study employed a scheme of Rg1 supplementation for female C57BL/6J mice, which started at the age of 12 months and ended at 24 months, to investigate the effects of Rg1 supplementation on the cognitive performance. We found that Rg1 supplementation improved the performance of aged mice in behavior test and significantly upregulated the expression of synaptic plasticity-associated proteins in hippocampus, including synaptophysin, N-methyl-d-aspartate receptor subunit 1, postsynaptic density-95, and calcium/calmodulin-dependent protein kinase II alpha, via promoting mammalian target of rapamycin pathway activation. These data provide further support for Rg1 treatment of cognitive degeneration during aging.

For aging individuals, age-related cognitive decline accompanying normal aging affects a variety of brain functions and greatly impairs their life quality. This aging process and related cognitive decline are not associated with significant neuron death (1) and produce a specific set of structural and functional synaptic alterations (2–5). Animal aging models report decreases in de novo protein synthesis (1), which has been well established as a requirement for synaptic plasticity and memory formation (6,7).

Mammalian target of rapamycin (mTOR), an evolutionarily conserved serine and threonine protein kinase, plays an important role in normal memory processes. It regulates the de novo synthesis of many synaptic plasticity-related proteins (8), including N-methyl-d-aspartate receptor subunit 1 (GluN1) (9), calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) (10), and postsynaptic density-95 (PSD-95) (11), via phosphorylation of its two downstream effectors p70 S6 kinase (p70S6K) and eIF4E-binding protein (4E-BP) (12). The former is a direct modulator of ribosomal protein S6 (13) whose phosphorylation state correlates with translation rates (14); the latter, 4E-BP2 in particular, the main isoform found in the brain (15), is involved in controlling the binding of the eukaryotic initiation factor 4E (eIF4E) to the initiation complex eIF4F, thus beginning translation (16). Pharmacological inhibition or genetic deletion of the mTOR pathway has been documented to result in learning and memory impairment in mammals (17), whereas enhancing neuronal mTOR signaling may improve memory function (18,19). These findings suggest that neuronal mTOR signaling pathway is strongly associated with cognitive performance of the brain and can serve as a research focus to gain insight into the pharmacological effects on cognitive decline of the aging individuals.

Ginseng, the root of Panax ginseng, has been used as an adaptogenic herb in traditional Chinese medicine for more than 2,000 years and is believed to promote the health of middle-aged and elderly people with a daily dose. It has been demonstrated to increase the body’s resistance to stress, trauma, anxiety, and fatigue (20). Ginsenosides, a class of steroidal glycosides, are the molecular components of ginseng responsible for these benefits (21). As one of the most abundant and active ingredients in P. ginseng (22), ginsenoside Rg1 (chemical structure shown in Figure 1) has been demonstrated in pharmacokinetic studies to pass through the blood–brain barrier and appear in cortex, hippocampus, and striatum corpora after oral administration to rats (23). It has been reported to produce protective effects against learning and memory impairment in several Alzheimer’s disease models including Aβ injected mice (24), SAMP8 (senescence-accelerated mouse prone 8) mice (25,26), and transgenic mAPP mice (27). However, the effects of Rg1 on cognitive decline during normal aging remain unknown, and the molecular mechanism of Rg1 action awaits further investigation.

Figure 1.

Chemical structure of ginsenoside Rg1. The two sugar side chains lie at C-6 and C-20.

Figure 1.

Chemical structure of ginsenoside Rg1. The two sugar side chains lie at C-6 and C-20.

The current study employed female C57BL/6J mice to look into the effect of long-term Rg1 supplementation on age-related cognition impairment, the structural alteration of synapses and the expression of several synaptic plasticity-associated proteins, and the role of aging and Rg1 supplementation in the activation of the mTOR pathway.

Materials and Methods

Chemical

Dehydrated Rg1 was obtained from Department of Biochemistry, Norman Bethune College of Medicine, Jilin University (Jilin, China) and arrived as a white powder of fine crystals with a melting point of 194°C–195°C, a molecular weight of 800, general formula C42H72O14, and 98% purity by reverse-phase high-pressure liquid chromatography. It was prepared as a 0.4mg/mL stock solution in saline, then aliquoted and stored frozen at −20°C.

The level of ginsenoside content in P. ginseng is usually 4%–7%, but can vary due to different seasons, cultivating soils, and extraction processes (28,29). The percentage of Rg1 in ginseng total saponins is approximately 7.6%.

Rg1 dosages were converted between adult human (60kg) and mouse (20g) by the body surface area normalization method, as described previously (30). The formula for dose translation is as follows: human equivalent dose (mg/kg) = animal dose (mg/kg) multiplied by animal Km/human Km. The values of Km factor, which is body weight (kg) divided by body surface area (m2), are 37 and 3 in human (60kg) and mouse (20g), respectively. Mice were given Rg1 at a dosage of 6 mg/kg every third day through an oral supplement. This dosage corresponds to 2–3g of ginseng per day in an adult human (60kg).

Mouse Care and Treatment Protocols

Female C57BL/6J mice were purchased from Department of Laboratory Animal Science of Peking University. All animals were born within a 2-week interval, housed five per cage in a pathogen-free colony (IVC system, Tecniplast, Italy), under a 12-hour dark/light cycle at 24°C, and allowed food and water ad libitum. All protocols and procedures used in these studies were approved by the Institutional Animal Care and Utilization Committee of Fujian Medical University.

Mice were randomly divided into Rg1-supplemented groups or aged control. In the former, 12-month-old mice were chronically supplemented with Rg1 by oral gavage; the latter mice received the same amount of saline for the same duration. Body weight and food intake were monitored every month during the experiment. Mice that were 4 and 12 months of age were used as young and middle-aged controls, respectively. All efforts were made to minimize animal suffering.

Behavioral Test

The Y-maze was made of Plexiglas, covered with white paper and consisted of three arms with an angle of 120° between each arm. Each arm was 8 cm wide × 30 cm long × 15 cm high. The floor of the maze was sprayed with alcohol after each individual trial to eliminate olfactory stimuli. Specific motifs were placed on the walls of each arm, thus allowing visual discrimination. Spontaneous alternation performance was tested as described previously (31,32). Each mouse was placed at the end of one arm and allowed to explore the apparatus for 5 minutes with the experimenter out of the animal’s sight. The sequence and total number of arms entered were recorded. Total activity was equivalent to the number of arm entries made by the mouse, whereas alternation opportunities were calculated by subtracting 2 from the total number of arms entered. Percentage alternation was calculated by dividing the number of triads containing entries into all three arms by alternation opportunities × 100. Novel object recognition test (33,34) consisted of two trials separated by an intertrial interval. The three arms were randomly designated: start arm, in which the mouse started to explore (always open); novel arm, which was blocked at the first trial but opened at the second trial; and other arm (always open). The first trial was 10 minutes in duration in which the mouse was allowed to explore only the start arm and other arm with the third arm (novel arm) blocked. After a 4-hour intertrial interval, the second trial was conducted. The mouse was placed back in the maze in the same starting arm, with free access to all three arms for 5 minutes. The number of entries and the time spent in each arm were recorded and analyzed. Data were expressed as percentage of performance in all three arms during the 5 minutes of test.

The Morris water maze was modified from Morris (35). Briefly, a black circular tank (120cm in diameter, 30cm in height) filled with 24°C ± 1°C water was divided to four equal quadrants by creating an imaginary “+.” Water was rendered opaque by adding milk. A black platform (diameter 10cm, height 24cm) submerged 1.0cm below the surface of the water was positioned in the middle of one of the quadrants and a red marker hung above the tank. Each mouse underwent four successive trials a day for 5 days in memory acquisition trials (training). The sequence of water entering positions differed daily, but the location of the platform was constant. These start positions were equal in length to the goal. Latency to find the platform was measured up to a maximum of 60 seconds. After locating the platform, the mouse was left there for 15 seconds prior to the next trial. If the mouse failed to locate the platform within 60 seconds, it was guided to the platform and allowed to stay there for 15 seconds. Latency and the average speed to reach the platform were recorded for each trial. On the sixth day, a “probe test” was performed to measure the strength of spatial memory retention, during which, mice were allowed to swim freely for 60 seconds in the pool without platform. There were two indexes calculated: the time (in seconds) spent by a mouse in the target quadrant in which the platform was hidden during acquisition trails; the number of times when mice exactly crossed over the previous position of the platform. Behavioral parameters were tracked and analyzed.

Transmission Electron Microscope

Mice were anesthetized with 10% chloral hydrate. After decapitation, the brains were rapidly removed and placed on ice. Hippocampus and CA1 were dissected out immediately. Tissue blocks were fixed by immersion in 2.5% buffered glutaraldehyde. Samples were then postfixed in 1% osmium tetroxide, dehydrated in ascending grades of ethanol, and embedded in Epon 812. Ultra microstructure of CA1 subregion in left hippocampus was observed under transmission electron microscope.

Immunohistochemistry

Animals were anesthetized with intraperitoneal injection of chloral hydrate. They were immediately perfused with 0.1 M phosphate-buffered saline (15 mL) and subsequently with 4% paraformaldhyde (20 mL). Brains were removed and postfixed in 4% paraformaldhyde for 4–6 hours and then transferred to 30% sucrose at 4°C. After the brains sank, they were flash frozen in liquid nitrogen and stored at −80°C until sectioning. Brains were sectioned into 30 μm slices on a freezing microtome (CM1850, Leica, Germany) and stored at −20°C in a cryoprotectant solution of 30% glycerin, 30% ethylene glycol (Sigma, MO), and 40% 0.1 M phosphate-buffered saline. After washes in 1× Tris-buffered saline (TBS), sections were treated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase and then washed in TBS. They were then blocked for 1 hour at room temperature in solution containing 0.3% Triton X-100, 0.25% body surface area, and 5% normal goat serum, followed by 24 hours incubation at 4°C in rabbit polyclonal antisera against either synaptophysin (Millipore, 1:10000) or GluN1 (Sigma, 1:200) in TBS containing 0.3% Triton X-100, 0.25% body surface area, and 2% normal goat serum. Sections were further incubated in biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) at 1:600 dilution for 90 minutes at room temperature. After being washed in TBS, the sections were incubated in Vector Elite avidin–peroxidase at 1:200 dilution for 60 minutes at room temperature. Finally, after a three-time wash in TBS, immunoreactivity was detected with diaminobenzidine. The sections were washed thoroughly in TBS, mounted onto polylysine-coated glass slides, air dried, dehydrated in ethanol, cleared in xylene, and then coverslipped with permanent mounting medium (Vector Laboratories).

Immunoblot Analysis

Mice were anesthetized with 10% chloral hydrate and perfused with 0.1 M phosphate-buffered saline (15 mL). Brains were rapidly removed and placed on ice. Hippocampi were dissected out immediately and quickly frozen in liquid nitrogen and stored at −80°C until lysis. Hippocampi were sonicated in cold lysis buffer with protease inhibitors (0.1M phosphate-buffered saline, 1% Triton X-100, 2.5mM Na4P2O5·10H2O, 2mM NaF, 1% protease inhibitor cocktail). After 10 minutes on ice, the samples were centrifuged at 16,000g for 15 minutes at 4°C. Supernatants were collected and a fraction of the total homogenates was removed to measure total protein concentrations. The concentration of each remaining homogenate was adjusted to 4.0mg/mL protein with lysis buffer and 6× sample buffer (125mM Tris, pH 6.8, 10% glycerol, 10% sodium dodecyl sulfate, 130mM dithiothreitol, and 0.006% bromophenol blue). The samples were boiled for 5 minutes, then aliquoted and stored at –20°C until Western blotting analysis. The total 60 μg protein lysate were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane. After blocked in a 5% nonfat dry milk solution in washing buffer (TBST, 10mM Tris, pH 7.6, 150mM NaCl, and 0.1% Tween-20), membranes were incubated overnight at 4°C with different antibodies: β-actin (Abcam, UK, 1:2000), GluN1 (Sigma, 1:1000), Anti-PSD-95 (Millipore, 1:1000), CaMKIIα (Cell Signaling Technology, 1:1000), synaptophysin (Millipore, 1:10000), phospho-mTOR (S2448; Cell Signaling Technology, 1:500), phospho-p70S6K (T389; Cell Signaling Technology, 1:400), phospho-4E-BP1 (Thr37/46; Cell Signaling Technology, 1:500), mTOR (Cell Signaling Technology, 1:500), p70S6K (Cell Signaling Technology, 1:500), and 4E-BP2 (Cell Signaling Technology, 1:500). After washed three times with TBST, membranes were incubated for 90 minutes with horseradish peroxidase-coupled secondary antibody (KPL, 1:2000) at room temperature. After washed three times with TBST, the resulting antigen–antibody–peroxidase complex was detected with an ECL kit (Millipore) and visualized by exposures of various lengths to Kodak film. The density of bands was quantified with Image J software. The amount of protein was expressed as a relative value to the levels of β-actin.

Statistical Analysis

Data from all procedures were expressed as “mean ± SEM.” Statistical analysis was performed with SPSS 11.0 software package and diagrams were plotted with Prism Graph Pad software. Indexes in acquisition trails such as escape latency and speed in Morris water maze test were analyzed with two-way analysis of variance of repeated measures. The data obtained from the “probe trial” in Morris water maze were analyzed with one-way analysis of variance, all other behavior tests and the expressions of proteins, with least significant difference test (equal variances assumed) for post hoc test or Dunnett’s T3 (equal variances not assumed) between groups and p < .05 was considered significant.

Results

Survival Conditions in Aged Control and Rg1-Supplemented Group

In the experiment, there were 33 and 32 mice in aged control and Rg1-supplemented group, respectively. Supplementation in mice with saline or Rg1 by oral gavage was initiated at 12 months of age and continued until 24 months. Mice were examined at least three times a week for signs of ill health and were allowed to die of natural causes. These death cases were almost due to tumor or severe weakness. Survival curves were plotted by the Kaplan–Meier method (Figure 2A). The survival time of Rg1-supplemented group was not different from that of the control group (χ2 = 0.311; p = .577). The tumor incidence rates of control group (27.27%) and Rg1-supplemented group (25.39%) were similar (Figure 2B). No excess mortality was observed in this stage of administration compared with that of other literatures, which involved the natural life span of C57BL/6J female mice (36–38).

Figure 2.

Survival conditions in aged control and Rg1-supplemented group. Supplementation in mice with saline or Rg1 by oral gavage was initiated at 12 months of age and continued until 24 months. (A) A Kaplan–Meier survival curve for mice is shown (n = 33 in control group and n = 32 in Rg1-supplemented group). (B) Tumor incidence rates in control group and Rg1-supplemented group. (C) Body weights and (D) food intakes were not statistically different between Rg1-supplemented mice (n = 5) and controls (n = 5).

Figure 2.

Survival conditions in aged control and Rg1-supplemented group. Supplementation in mice with saline or Rg1 by oral gavage was initiated at 12 months of age and continued until 24 months. (A) A Kaplan–Meier survival curve for mice is shown (n = 33 in control group and n = 32 in Rg1-supplemented group). (B) Tumor incidence rates in control group and Rg1-supplemented group. (C) Body weights and (D) food intakes were not statistically different between Rg1-supplemented mice (n = 5) and controls (n = 5).

To exclude the possibility that long-term intragastric administration might injure the esophagogastric mucosa of mice and further influence the feeding and body condition, we measured food consumption and body weight every month in a random subset of mice (n = 5 in each group). The body weight (Figure 2C) and food intake (Figure 2D) did not decrease and were not statistically different between the two groups, suggesting that the living status in mice was not severely influenced by the gavage administration. The physical ability of mice had also been checked before behavioral tests and those with mobility disabilities and cataracts were eliminated.

Rg1 Improves Performance in Spatial Working and Memory Formation

In order to evaluate the effect of Rg1 supplementation on improving memory impairments in aged mice, we tested the mice with Y-maze that represents a measure of spatial working memory (39) (Figure 3A–D). Notably, compared with young and middle-aged controls, aged mice showed significantly reduced levels of spontaneous alternation performance in the Y-maze. This decline was rescued by Rg1 supplementation (F(3,28) = 5.310, p < .01; Figure 3B). Furthermore, the total number of arm entries during Y-maze testing was not significantly different between the four groups (F(3,28) = 0.676, p = .574; Figure 3A). Therefore, levels of exploratory activity were not affected in these mice. Novel object recognition is based on the premise that mice will explore a novel object more than a familiar one provided the animal is able to remember the familiar object. Statistical analysis showed the percentage of number of visits (F(3,28) = 7.00, p < .01; Figure 3C) and time spent (F(3,28) = 6.095, p < .01; Figure 3D) in the novel arm decreased significantly in the aged mice in comparison with young controls. However, Rg1-supplemented mice showed improved performance. These results indicate that Rg1-supplemented mice display improved performance in spatial working memory.

Figure 3.

Rg1 improved age-related decline in spatial working memory as indicated by Y-maze. Spatial working memory of young (4 months, n = 8), middle-aged (12 months, n = 8), aged (24 months, n = 8), and Rg1-supplemented (24 months, n = 8) mice was evaluated using spontaneous alternation (A and B) and novel object recognition (C and D). (A and B) No difference of the total number of arm entries reflecting exploratory activities of mice was observed among the four groups based on spontaneous alternation test. The levels of spontaneous alternation performance in aged mice were significantly reduced compared with that of young (p < .01) and middle-aged controls (p < .01). The level in Rg1-supplemented mice was rescued (p < .01). (C and D) In novel object recognition, entries and time spent in each arm for mice visiting the novel, start, and other arms were recorded. Aged mice showed less novel arm exploratory compared with the young (p < .01 in entries and time) and middle-aged controls (p < .01 in entries and p < .05 in time). Rg1-supplemented mice showed improved performance (p < .05 in entries and time). Rg1 reduced age-related decline in spatial learning and memory as indicated by the Morris water maze (EH). Young (4 months, n = 8), middle-aged (12 months, n = 8), aged (24 months, n = 8), and Rg1-supplemented (24 months, n = 8) mice were tested on components of the Morris water maze. (E) In learning trials, escape latencies per group of four trials were tested more than 5 days. The latency in aged mice was significantly longer than that of young (p < .001) and middle-aged mice (p < .01). The latency in Rg1-supplemented mice was significantly shorter than that of aged controls (p < .05). (F) This graph shows the swimming speed data in learning trials. The swimming speeds of each group did not differ on the training day. (G) Probe trial performance concerning retention of the general location of platform is shown by depicting time spent in the target quadrant where the submerged platform had been located. The young, middle-aged, and Rg1-supplemented groups spent significantly more time in the target quadrant compared with the aged mice (p < .001, .05, and .05, respectively) in the spatial probe trial. (H) The graph shows retention performance concerning the number of times the mice crossed over the exact location of submerged platform in the probe trials. The young, middle-aged, and Rg1-supplemented groups exhibited significantly more platform crosses than the aged mice (p < .001, .05, and .01, respectively). *p < .05, **p < .01, ***p < .001 compared with the aged control group.

Figure 3.

Rg1 improved age-related decline in spatial working memory as indicated by Y-maze. Spatial working memory of young (4 months, n = 8), middle-aged (12 months, n = 8), aged (24 months, n = 8), and Rg1-supplemented (24 months, n = 8) mice was evaluated using spontaneous alternation (A and B) and novel object recognition (C and D). (A and B) No difference of the total number of arm entries reflecting exploratory activities of mice was observed among the four groups based on spontaneous alternation test. The levels of spontaneous alternation performance in aged mice were significantly reduced compared with that of young (p < .01) and middle-aged controls (p < .01). The level in Rg1-supplemented mice was rescued (p < .01). (C and D) In novel object recognition, entries and time spent in each arm for mice visiting the novel, start, and other arms were recorded. Aged mice showed less novel arm exploratory compared with the young (p < .01 in entries and time) and middle-aged controls (p < .01 in entries and p < .05 in time). Rg1-supplemented mice showed improved performance (p < .05 in entries and time). Rg1 reduced age-related decline in spatial learning and memory as indicated by the Morris water maze (EH). Young (4 months, n = 8), middle-aged (12 months, n = 8), aged (24 months, n = 8), and Rg1-supplemented (24 months, n = 8) mice were tested on components of the Morris water maze. (E) In learning trials, escape latencies per group of four trials were tested more than 5 days. The latency in aged mice was significantly longer than that of young (p < .001) and middle-aged mice (p < .01). The latency in Rg1-supplemented mice was significantly shorter than that of aged controls (p < .05). (F) This graph shows the swimming speed data in learning trials. The swimming speeds of each group did not differ on the training day. (G) Probe trial performance concerning retention of the general location of platform is shown by depicting time spent in the target quadrant where the submerged platform had been located. The young, middle-aged, and Rg1-supplemented groups spent significantly more time in the target quadrant compared with the aged mice (p < .001, .05, and .05, respectively) in the spatial probe trial. (H) The graph shows retention performance concerning the number of times the mice crossed over the exact location of submerged platform in the probe trials. The young, middle-aged, and Rg1-supplemented groups exhibited significantly more platform crosses than the aged mice (p < .001, .05, and .01, respectively). *p < .05, **p < .01, ***p < .001 compared with the aged control group.

To further determine the effect of Rg1 on improving spatial learning and memory impairments in normal aged mice, we examined the performance of each group in the Morris water maze test. This test challenged their ability to learn the location of the hidden platform by relevant visual cues. In this test, mice were trained in the water maze for 5 days. The escape latency in aged mice was longer than that of young and middle-aged controls (Figure 3E). Mice supplemented with Rg1 showed a significant decrease in escape latency compared with the aged control mice, but did not differ from the middle-aged controls. The main effect of day was statistically significant (F(4,112) = 28.449, p < .001). The main effect of group was also statistically significant (F(3,28) = 8.261, p < .001). The Day × Group interaction was significant (F(12,112) = 2.95, p = .001). Two-way repeated measures analysis of variance revealed a significant increase in escape latency in the aged mice in comparison with young controls and middle-aged controls (F(1,14) = 33.08, p < .001; F(1,14) = 20.139, p < .01, respectively) and significant decreases in Rg1-supplemented mice compared with aged control mice (F(1,14) = 6.396, p < .05). However, in the four groups, swimming speeds did not differ in the training day (day: F(4,112) = 4.287, p = .003; group: F(3,28) = 2.356, p > .05; or Day × Group: F(12,112) = 0.436, p > .05; Figure 3F). In the probe trial, with the platform removed, the time-aged mice spent in the target quadrant (Figure 3G) were significantly shorter than that of young and middle-aged controls (F(3,28) = 8.898, p < .001). The number of crosses over the position (Figure 3H) where the platform had been located also decreased significantly (F(3,28) = 6.992, p < .01). Rg1-supplemented mice spent more time in the target quadrant and crossing numbers were significantly increased over that of aged control mice. These results indicate that Rg1-supplemented mice display improved performance in learning and memory assays.

Rg1 Alleviates Age-Related Synapse Structural Alterations Observed by Transmission Electron Microscopy

The synapse in the nervous system consists of three elements. The pre- and postsynaptic membranes appear parallel to each other and are separated by a synaptic cleft (40). In the chemical synapses of young and middle-aged mice, the typical structure was observed under transmission electron microscope (Figure 4A and B). Briefly, plenty of synaptic vesicles, which contain various neurotransmitters, were observed inside the presynaptic membrane. PSD was concentrated on the intracellular surface of opposing postsynaptic membrane. The synaptic cleft appeared as a narrow gap with rigidly paralleled membranes. In aged-mice (Figure 4C), the structure of synapses was damaged compared with that of the young group, with deformed anterior region, decreased synaptic vesicles, reduced postsynaptic region, and a decreased PSD area. Postsynaptic membrane swelled and synaptic cleft obviously widened. In Rg1-supplemented group (Figure 4D), the damage of synaptic structure was alleviated to some degree, with more regular synaptic form, more vesicles evenly distributed in the presynaptic area, a slightly thickened postsynaptic membrane, and an identifiable synaptic cleft. These results indicate that Rg1 supplementation protects the synaptic structure of CA1 in aged mice.

Figure 4.

Rg1 treatment alleviates age-dependent synaptic structure degradation. Observed by transmission electron microscope, the chemical synapses in young mice (A) and middle-aged mice (B) showed the typical structure: plenty of synaptic vesicles were inside of the presynaptic membrane. Postsynaptic density was concentrated on the intracellular surface of opposing postsynaptic membrane. The synaptic cleft appeared as a narrow gap with rigidly paralleled membranes. (C). The structure of synapses in aged group was damaged compared with that of the young group, with deformed anterior region, decreased synaptic vesicles, reduced postsynaptic region, and a decreased postsynaptic density area. Postsynaptic membrane swelled and synaptic cleft obviously widened. (D). In Rg1-supplemented group, this damage of synaptic structure was alleviated to some degree, with more regular synaptic form, more evenly distributed vesicles in presynaptic area, slightly thickened postsynaptic membrane, and identifiable synaptic cleft. The black arrow indicates the synapse. The arrowheads indicate synaptic vesicles. Scale bar = 300 nm.

Figure 4.

Rg1 treatment alleviates age-dependent synaptic structure degradation. Observed by transmission electron microscope, the chemical synapses in young mice (A) and middle-aged mice (B) showed the typical structure: plenty of synaptic vesicles were inside of the presynaptic membrane. Postsynaptic density was concentrated on the intracellular surface of opposing postsynaptic membrane. The synaptic cleft appeared as a narrow gap with rigidly paralleled membranes. (C). The structure of synapses in aged group was damaged compared with that of the young group, with deformed anterior region, decreased synaptic vesicles, reduced postsynaptic region, and a decreased postsynaptic density area. Postsynaptic membrane swelled and synaptic cleft obviously widened. (D). In Rg1-supplemented group, this damage of synaptic structure was alleviated to some degree, with more regular synaptic form, more evenly distributed vesicles in presynaptic area, slightly thickened postsynaptic membrane, and identifiable synaptic cleft. The black arrow indicates the synapse. The arrowheads indicate synaptic vesicles. Scale bar = 300 nm.

Rg1 Upregulates the Expression of Synaptic Plasticity-Related Proteins

Synaptic plasticity is a key component of the learning machinery in the brain. Two synaptic plasticity-related proteins, synaptophysin and GluN1, were analyzed by immunohistochemistry. We observed age-dependent decreases in the expression of both synaptic markers in the CA1 region (Figure 5A). The expression of the pre- and postsynaptic proteins, including synaptophysin, GluN1, PSD-95, and CaMKIIα, was measured by immunoblotting hippocampal lysates (Figure 5B). The expression of synaptophysin was significantly reduced in the aged controls compared with that of young mice (F(3,20) = 12.803, p < .001) and significantly increased in Rg1-supplemented mice compared with that of the aged controls (p < .01). Similarly, the expressions of GluN1, PSD-95, and CaMKIIα were significantly lowered in aged mice when that of compared with young and middle-aged mice (F(3,20) = 23.521, p < .001 for GluN1; F(3,20) = 37.403, p < .001 for CaMKIIα; F(3,20) = 13.963, p < 0.001 for PSD-95). The expressions of these markers were increased in mice supplemented with Rg1 in comparison with aged controls (p < .05, p < .001, and p < .05, respectively). These results indicate that the expression of these synaptic plasticity-related proteins decreases significantly in an age-related manner and can be upregulated by Rg1 supplementation.

Figure 5.

Rg1 upregulates the expression of synapse plasticity related proteins. (A) Synaptophysin and N-methyl-d-aspartate receptor subunit 1 (GluN1) are shown by immunohistochemistry in the CA1 region of the hippocampus. There was an age-dependent decrease in the expression of each synaptic marker. Rg1-supplemented mice were not grossly different from middle aged mice. Scale bar = 100 μm. (B) The expression of synaptophysin, GluN1, postsynaptic density-95 (PSD-95), and calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) in hippocampus (n = 6 per group) was assayed by Western blotting. β-Actin was used as a control of protein loading. The bands of Western blot were scanned, and the ratios of optical density of specific bands and β-actin were illustrated. *p < .05, **p < .01, ***p < .001 compared with the aged control group.

Figure 5.

Rg1 upregulates the expression of synapse plasticity related proteins. (A) Synaptophysin and N-methyl-d-aspartate receptor subunit 1 (GluN1) are shown by immunohistochemistry in the CA1 region of the hippocampus. There was an age-dependent decrease in the expression of each synaptic marker. Rg1-supplemented mice were not grossly different from middle aged mice. Scale bar = 100 μm. (B) The expression of synaptophysin, GluN1, postsynaptic density-95 (PSD-95), and calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) in hippocampus (n = 6 per group) was assayed by Western blotting. β-Actin was used as a control of protein loading. The bands of Western blot were scanned, and the ratios of optical density of specific bands and β-actin were illustrated. *p < .05, **p < .01, ***p < .001 compared with the aged control group.

Rg1 Promotes the Activation of mTOR Signaling Pathway

mTOR signaling modulates synaptic plasticity by regulating protein translation. Therefore, we investigated whether Rg1 supplementation would affect mTOR signaling in the hippocampus. We used immunoblotting to determine the phosphorylation state of mTOR/p70S6K/4E-BP2 (Figure 6). The phosphorylation of mTOR (S2448) and p70S6K (T389) decreased significantly in the aged group compared with that of young and middle-aged controls (F(3,20) = 13.768, p < .001 for p-mTOR; F(3,20) =9.257, p < .01 for p-p70S6K). 4E-BP1 (Thr37/46) also showed a moderate, statistically insignificant decrease (F(3,12) = 1.637, p = .233). However, total protein levels were not changed between groups. Notably, the phosphorylation of mTOR, p70S6K, and 4E-BP1 was increased in Rg1-supplemented mice compared with that of aged controls. Thus, the activation of the mTOR signaling pathway was increased in Rg1-supplemented mice.

Figure 6.

Rg1 promotes activation of the mammalian target of rapamycin (mTOR) signaling pathway. phospho-mTOR/mTOR, phospho-p70S6K/p70S6K, phospho-4E-BP1 (Thr37/46)/4E-BP2 were assayed by Western blotting (n = 6 per group). Phospho-4E-BP1 (Thr37/46) is known to cross-react with 4E-BP2 and 4E-BP3 at analogous phosphorylation sites. β-Actin was used as a control of protein loading. The bands of Western blot were scanned, and the ratios of optical density of specific bands and β-actin were illustrated. *p < .05, **p < .01, ***p < .001 compared with the aged control group.

Figure 6.

Rg1 promotes activation of the mammalian target of rapamycin (mTOR) signaling pathway. phospho-mTOR/mTOR, phospho-p70S6K/p70S6K, phospho-4E-BP1 (Thr37/46)/4E-BP2 were assayed by Western blotting (n = 6 per group). Phospho-4E-BP1 (Thr37/46) is known to cross-react with 4E-BP2 and 4E-BP3 at analogous phosphorylation sites. β-Actin was used as a control of protein loading. The bands of Western blot were scanned, and the ratios of optical density of specific bands and β-actin were illustrated. *p < .05, **p < .01, ***p < .001 compared with the aged control group.

Discussion

The current study demonstrated that age-related cognitive decline was accompanied by structural alteration of synapses and downregulation of synaptic plasticity-associated proteins in the hippocampus. We found that long-term Rg1 supplementation in mice could alleviate age-related cognitive decline. In addition, the mTOR signaling pathway in the hippocampus, which upregulates the synthesis of synaptic plasticity-associated proteins, was activated by Rg1 supplementation.

In current study, the scheme of ginsenoside Rg1 supplementation for female C57BL/6J mice was initiated when the mice were at 12 months of age and terminated at 24 months. The time span involved is substantiated by proven evidence. Jackson Laboratory considers 10–14 months to be “middle age” for a C57BL/6J mouse, a rough equivalent of 38–47 years in humans. Accordingly, old age begins at 18–24 months and some studies reported that 24–25 months was approximately the median life span of C57BL/6J mice. The course of Rg1 supplementation in this study is analogous to individuals beginning ginseng supplements in midlife. The decision on female mice is also based on solid findings. Compared with males, human females have a longer life expectancy and studies have reported them to have higher incidence of age-related cognitive decline (41,42). Recent evidence suggests that age-related changes in certain regions of the brain occur earlier in females than in males, which are closely associated with environmental insults including psychological stress and trauma (43). For these reasons, naturally aged female mice were used as experimental subjects in this study. We observed significant age-related spatial learning and memory formation impairment in older mice, being consistent with that of previous reports (44,45). Of note, we found that long-term, low-dose Rg1 supplementation improved these memory impairments in aged mice. Meanwhile, no significant difference in survival time, tumor incidence, weight, and food intake was observed between Rg1-supplemented group and the control group. It suggests that the dosage of Rg1 supplementation used in this study was safe and recommendable.

The morphological changes of synapse correspond to the decrease in overall synaptic function (46). Here, we observed that age-dependent changes in synaptic ultrastructure in the hippocampal CA1 area of C57BL/6J mice and these changes were alleviated by Rg1 treatment in aged mice. Therefore, we further examined synaptic plasticity-associated proteins. Synaptophysin is a glycoprotein involved in synaptic vesicle trafficking, docking, and fusion leading to neurotransmitter secretion (47,48). Alterations in synaptophysin may affect the size and number of presynaptic vesicles. N-methyl-d-aspartic acid receptor (NMDAR) is a member of the ionotropic glutamate receptor family that is best known for its role in long-term potentiation. GluN1 is a subunit required for receptor function and formation (49). PSD-95 localized in the postsynaptic density recruits and clusters NMDA receptors, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) pro panoic acid (AMPA) receptors, cytoskeletal components, and signal transduction molecules in response to synaptic activity (50,51). Transgenic deletion of PSD-95 causes severe spatial learning and memory impairment (52). CaMKIIα exists in both pre- and postsynaptic locations. In presynaptic elements, it is involved in the mobilization of synaptic vesicle clusters (53) and regulation of neurotransmitter release (54). In terms of its postsynaptic role, it regulates NMDA receptor expression and localization to the postsynaptic membrane (55). Animals with decreased CaMKIIα activity showed impaired synaptic plasticity and memory formation (56,57). In this study, we have identified significant age-related decreases in hippocampal expression of four proteins with extensive roles in synaptic plasticity. As described earlier, aged C57BL/6J mice, whose plasticity-associated proteins were downregulated, displayed poor learning and memory formation in behavior tests. Treatment with Rg1 improved memory performance and alleviated the reduction of plasticity-associated proteins in aged mice. These results suggest that improvement in learning and memory formation by Rg1 treatment may be associated with the upregulation of synaptic plasticity-associated proteins.

The mTOR signaling pathway is of critical importance in long-term synaptic plasticity and memory storage. It contributes to synaptic plasticity by regulating protein synthesis. Increased formation of mTOR complex 1 (mTORC1) is involved in the activation of S6K and repression of the 4E-BPs (58,59). The activation of the pathway can be inhibited pharmacologically and genetically. Previous studies have shown that rapamycin, a drug that selectively inhibits mTORC1 formation, blocked long-term memory in mammals (60–63). Mice with genetic mutations in 4E-BP2 (15,64), S6K1, and S6K2 (65) presented a number of learning and memory impairments. Particularly, Stoica and colleagues (66) have successfully developed an innovative method based on the synergistic action of genetics and pharmacology to specifically and directly inhibit mTORC1 in the brain. The pharmacological or genetic inhibition of mTORC1 blocks L-long-term potentiation and impairs long-term memory reconsolidation in mice. On the other hand, the enhancement of the mTOR signaling can also promote memory function. A recent study documents that FKBP12 knockout enhanced neuronal mTOR signaling, thus improving memory performance in mice (19). More studies report that the activation of mTOR signaling pathway in dorsal hippocampus by estrogen and progesterone, respectively, can enhance memory consolidation in female mice (67,68). Taken together, these findings suggest that the mTOR pathway can serve as a research window to gain insight into its role in long-lasting synaptic plasticity and memory processes.

Normal aging is accompanied by reduced memory function. Studies with animal models demonstrated that signaling pathways that support memory formation were altered in the aged brain (69,70). We show in our study that the memory decline in aged animals is probably related to the decrease of mTOR activation in hippocampus. This finding can be further supported by the studies of Deli and colleagues and Ma and colleagues. The former found that when mTORC1 activity was blocked, the young C57BL/6J mice displayed impaired spatial memory retrieval (71); the latter reported that downregulation of mTOR signaling by Aβ toxicity also induced synaptic dysfunction (72). Another finding in our study is that the decreased phosphorylation of mTOR signaling molecules was alleviated by Rg1 supplementation. Herein, we suggest that long-term Rg1 supplementation can promote the activation of the mTOR pathway and improve age-related memory decline.

Contradictory to our findings, there are studies showing that aging-associated neurodegenerative diseases may benefit from reducing mTOR activity. For example, rapamycin treatment can prophylactically combat Alzheimer’s disease (73,74) and Parkinson’s disease (75,76). Two recent reports (77,78) demonstrated that lifelong rapamycin administration ameliorates age-dependent cognitive degeneration. Rapamycin binds FKBP12, preventing mTOR complex formation, slowing translation of new proteins, and inducing autophagy (79). As aging-associated diseases are connected with accumulation of misfolded or aggregated proteins, it is possible that decreasing the rate of protein synthesis may increase removal of misfolded or damaged proteins. Furthermore, induction of autophagy by rapamycin can clear damaged organelles and proteins that are resistant to degradation by the proteosome (8). Therefore, we speculate that a balanced regulation of mTOR signaling, rather than a simplified switching on or off of this pathway, might be a better way to treat age-related neurological disorders.

In conclusion, during normal aging, age-related cognitive decline is accompanied by downregulation of mTOR signaling in the hippocampus. Long-term Rg1 treatment can greatly improve cognitive performance by enhancing the activation of mTOR signaling and upregulating synaptic plasticity-related protein synthesis. These findings suggest ginsenoside Rg1 can serve as a candidate for alleviating age-related cognitive decline.

Funding

This work was supported by grant from the National Basic Research Program of China (2007CB507400).

References

1.
Burke
SN
Barnes
CA
.
Neural plasticity in the ageing brain
.
Nat Rev Neurosci
 .
2006
;
7
:
30
40
.
2.
Peters
A
Sethares
C
Luebke
JI
.
Synapses are lost during aging in the primate prefrontal cortex
.
Neuroscience
 .
2008
;
152
:
970
981
.
3.
Soghomonian
JJ
Sethares
C
Peters
A
.
Effects of age on axon terminals forming axosomatic and axodendritic inhibitory synapses in prefrontal cortex
.
Neuroscience
 .
2010
;
168
:
74
81
.
4.
Peters
A
Kemper
T
.
A review of the structural alterations in the cerebral hemispheres of the aging rhesus monkey
.
Neurobiol Aging
 .
2012
;
33
:
2357
2372
.
5.
VanGuilder
HD
Farley
JA
Yan
H
et al. 
Hippocampal dysregulation of synaptic plasticity-associated proteins with age-related cognitive decline
.
Neurobiol Dis
 .
2011
;
43
:
201
212
.
6.
Squire
LR
Davis
HP
.
The pharmacology of memory: a neurobiological perspective
.
Annu Rev Pharmacol Toxicol
 .
1981
;
21
:
323
356
.
7.
Kelleher
RJ
III
Govindarajan
A
Tonegawa
S
.
Translational regulatory mechanisms in persistent forms of synaptic plasticity
.
Neuron
 .
2004
;
44
:
59
73
.
8.
Garelick
MG
Kennedy
BK
.
TOR on the brain
.
Exp Gerontol
 .
2011
;
46
:
155
163
.
9.
Schratt
GM
Nigh
EA
Chen
WG
Hu
L
Greenberg
ME
.
BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development
.
J Neurosci
 .
2004
;
24
:
7366
7377
.
10.
Gong
R
Park
CS
Abbassi
NR
Tang
SJ
.
Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons
.
J Biol Chem
 .
2006
;
281
:
18802
18815
.
11.
Lee
CC
Huang
CC
Wu
MY
Hsu
KS
.
Insulin stimulates postsynaptic density-95 protein translation via the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway
.
J Biol Chem
 .
2005
;
280
:
18543
18550
.
12.
Burnett
PE
Barrow
RK
Cohen
NA
Snyder
SH
Sabatini
DM
.
RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1
.
Proc Natl Acad Sci U S A
 .
1998
;
95
:
1432
1437
.
13.
Meyuhas
O
.
Physiological roles of ribosomal protein S6: one of its kind
.
Int Rev Cell Mol Biol
 .
2008
;
268
:
1
37
.
14.
Ferrari
S
Thomas
G
.
S6 phosphorylation and the p70s6k/p85s6k
.
Crit Rev Biochem Mol Biol
 .
1994
;
29
:
385
413
.
15.
Banko
JL
Poulin
F
Hou
L
DeMaria
CT
Sonenberg
N
Klann
E
.
The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus
.
J Neurosci
 .
2005
;
25
:
9581
9590
.
16.
Costa-Mattioli
M
Sossin
WS
Klann
E
Sonenberg
N
.
Translational control of long-lasting synaptic plasticity and memory
.
Neuron
 .
2009
;
61
:
10
26
.
17.
Hoeffer
CA
Klann
E
.
mTOR signaling: at the crossroads of plasticity, memory and disease
.
Trends Neurosci
 .
2010
;
33
:
67
75
.
18.
Dash
PK
Orsi
SA
Moore
AN
.
Spatial memory formation and memory-enhancing effect of glucose involves activation of the tuberous sclerosis complex-Mammalian target of rapamycin pathway
.
J Neurosci
 .
2006
;
26
:
8048
8056
.
19.
Hoeffer
CA
Tang
W
Wong
H
et al. 
Removal of FKBP12 enhances mTOR-Raptor interactions, LTP, memory, and perseverative/repetitive behavior
.
Neuron
 .
2008
;
60
:
832
845
.
20.
Winston
D
Maimes
S
.
Adaptogens: Herbs for Strength, Stamina, and Stress
 .
Rochester, VT
:
Healing Arts Press
;
2007
.
21.
Liu
CX
Xiao
PG
.
Recent advances on ginseng research in China
.
J Ethnopharmacol
 .
1992
;
36
:
27
38
.
22.
Radad
K
Gille
G
Moldzio
R
Saito
H
Rausch
WD
.
Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate
.
Brain Res
 .
2004
;
1021
:
41
53
.
23.
Zhang
JT
Chui
DH
Chen
CF
Liu
GZ
.
The Chemistry, Metabolism and Biological Activities of Ginseng
 .
Beijing, China
:
Chemical Industry Press
;
2006
24.
Tohda
C
Matsumoto
N
Zou
K
Meselhy
MR
Komatsu
K
.
Abeta(25-35)-induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by M1, A metabolite of protopanaxadiol-type saponins
.
Neuropsychopharmacology
 .
2004
;
29
:
860
868
.
25.
Shi
YQ
Huang
TW
Chen
LM
et al. 
Ginsenoside Rg1 attenuates amyloid-beta content, regulates PKA/CREB activity, and improves cognitive performance in SAMP8 mice
.
J Alzheimers Dis
 .
2010
;
19
:
977
989
.
26.
Zhao
H
Li
Q
Zhang
Z
Pei
X
Wang
J
Li
Y
.
Long-term ginsenoside consumption prevents memory loss in aged SAMP8 mice by decreasing oxidative stress and up-regulating the plasticity-related proteins in hippocampus
.
Brain Res
 .
2009
;
1256
:
111
122
.
27.
Fang
F
Chen
X
Huang
T
Lue
LF
Luddy
JS
Yan
SS
.
Multi-faced neuroprotective effects of Ginsenoside Rg1 in an Alzheimer mouse model
.
Biochim Biophys Acta
 .
2012
;
1822
:
286
292
.
28.
Sievenpiper
JL
Arnason
JT
Leiter
LA
Vuksan
V
.
Variable effects of American ginseng: a batch of American ginseng (Panax quinquefolius L.) with a depressed ginsenoside profile does not affect postprandial glycemia
.
Eur J Clin Nutr
 .
2003
;
57
:
243
248
.
29.
Um
JY
Chung
HS
Kim
MS
et al. 
Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP
.
Biol Pharm Bull
 .
2001
;
24
:
872
875
.
30.
Reagan-Shaw
S
Nihal
M
Ahmad
N
.
Dose translation from animal to human studies revisited
.
FASEB J
 .
2008
;
22
:
659
661
.
31.
Ohno
M
Sametsky
EA
Younkin
LH
et al. 
BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer’s disease
.
Neuron
 .
2004
;
41
:
27
33
.
32.
Kimura
R
Devi
L
Ohno
M
.
Partial reduction of BACE1 improves synaptic plasticity, recent and remote memories in Alzheimer’s disease transgenic mice
.
J Neurochem
 .
2010
;
113
:
248
261
.
33.
Ma
MX
Chen
YM
He
J
Zeng
T
Wang
JH
.
Effects of morphine and its withdrawal on Y-maze spatial recognition memory in mice
.
Neuroscience
 .
2007
;
147
:
1059
1065
.
34.
Zhang
J
He
J
Chen
YM
Wang
JH
Ma
YY
.
Morphine and propranolol co-administration impair consolidation of Y-maze spatial recognition memory
.
Brain Res
 .
2008
;
1230
:
150
157
.
35.
Vorhees
CV
Williams
MT
.
Morris water maze: procedures for assessing spatial and related forms of learning and memory
.
Nat Protoc
 .
2006
;
1
:
848
858
.
36.
Quick
KL
Ali
SS
Arch
R
Xiong
C
Wozniak
D
Dugan
LL
.
A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice
.
Neurobiol Aging
 .
2008
;
29
:
117
128
.
37.
Ko
KM
Chiu
PY
Leung
HY
et al. 
Long-term dietary supplementation with a yang-invigorating Chinese herbal formula increases lifespan and mitigates age-associated declines in mitochondrial antioxidant status and functional ability of various tissues in male and female C57BL/6J mice
.
Rejuvenation Res
 .
2010
;
13
:
168
171
.
38.
Turturro
A
Duffy
P
Hass
B
Kodell
R
Hart
R
.
Survival characteristics and age-adjusted disease incidences in C57BL/6 mice fed a commonly used cereal-based diet modulated by dietary restriction
.
J Gerontol A Biol Sci Med Sci
 .
2002
;
57
:
B379
B389
.
39.
Lalonde
R
.
The neurobiological basis of spontaneous alternation
.
Neurosci Biobehav Rev
 .
2002
;
26
:
91
104
.
40.
Hernandez-Nicaise
ML
.
The nervous system of ctenophores. III. Ultrastructure of synapses
.
J Neurocytol
 .
1973
;
2
:
249
263
.
41.
von Strauss
E
Viitanen
M
De Ronchi
D
Winblad
B
Fratiglioni
L
.
Aging and the occurrence of dementia: findings from a population-based cohort with a large sample of nonagenarians
.
Arch Neurol
 .
1999
;
56
:
587
592
.
42.
Corrada
MM
Brookmeyer
R
Berlau
D
Paganini-Hill
A
Kawas
CH
.
Prevalence of dementia after age 90: results from the 90+ study
.
Neurology
 .
2008
;
71
:
337
343
.
43.
Yuan
Y
Chen
YP
Boyd-Kirkup
J
Khaitovich
P
Somel
M
.
Accelerated aging-related transcriptome changes in the female prefrontal cortex
.
Aging Cell
 .
2012
;
11
:
894
901
.
44.
Benice
TS
Rizk
A
Kohama
S
Pfankuch
T
Raber
J
.
Sex-differences in age-related cognitive decline in C57BL/6J mice associated with increased brain microtubule-associated protein 2 and synaptophysin immunoreactivity
.
Neuroscience
 .
2006
;
137
:
413
423
.
45.
Yu
YF
Zhai
F
Dai
CF
Hu
JJ
.
The relationship between age-related hearing loss and synaptic changes in the hippocampus of C57BL/6J mice
.
Exp Gerontol
 .
2011
;
46
:
716
722
.
46.
Adams
MM
Shi
L
Linville
MC
et al. 
Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability
.
Exp Neurol
 .
2008
;
211
:
141
149
.
47.
Fuentes-Santamaría
V
Alvarado
JC
Henkel
CK
Brunso-Bechtold
JK
.
Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus
.
Neuroscience
 .
2007
;
148
:
1033
1047
.
48.
Südhof
TC
.
The synaptic vesicle cycle: a cascade of protein-protein interactions
.
Nature
 .
1995
;
375
:
645
653
.
49.
Lau
GC
Saha
S
Faris
R
Russek
SJ
.
Up-regulation of NMDAR1 subunit gene expression in cortical neurons via a PKA-dependent pathway
.
J Neurochem
 .
2004
;
88
:
564
575
.
50.
Béïque
JC
Lin
DT
Kang
MG
Aizawa
H
Takamiya
K
Huganir
RL
.
Synapse-specific regulation of AMPA receptor function by PSD-95
.
Proc Natl Acad Sci U S A
 .
2006
;
103
:
19535
19540
.
51.
Vickers
CA
Stephens
B
Bowen
J
Arbuthnott
GW
Grant
SG
Ingham
CA
.
Neurone specific regulation of dendritic spines in vivo by post synaptic density 95 protein (PSD-95)
.
Brain Res
 .
2006
;
1090
:
89
98
.
52.
Migaud
M
Charlesworth
P
Dempster
M
et al. 
Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein
.
Nature
 .
1998
;
396
:
433
439
.
53.
Tao-Cheng
JH
Dosemeci
A
Winters
CA
Reese
TS
.
Changes in the distribution of calcium calmodulin-dependent protein kinase II at the presynaptic bouton after depolarization
.
Brain Cell Biol
 .
2006
;
35
:
117
124
.
54.
Nielander
HB
Onofri
F
Valtorta
F
et al. 
Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases
.
J Neurochem
 .
1995
;
65
:
1712
1720
.
55.
Park
CS
Elgersma
Y
Grant
SG
Morrison
JH
.
alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus
.
Neuroscience
 .
2008
;
151
:
43
55
.
56.
Elgersma
Y
Sweatt
JD
Giese
KP
.
Mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase II function in plasticity and cognition
.
J Neurosci
 .
2004
;
24
:
8410
8415
.
57.
Miller
S
Yasuda
M
Coats
JK
Jones
Y
Martone
ME
Mayford
M
.
Disruption of dendritic translation of CaMKIIalpha impairs stabilization of synaptic plasticity and memory consolidation
.
Neuron
 .
2002
;
36
:
507
519
.
58.
Klann
E
Dever
TE
.
Biochemical mechanisms for translational regulation in synaptic plasticity
.
Nat Rev Neurosci
 .
2004
;
5
:
931
942
.
59.
Guertin
DA
Sabatini
DM
.
An expanding role for mTOR in cancer
.
Trends Mol Med
 .
2005
;
11
:
353
361
.
60.
Tischmeyer
W
Schicknick
H
Kraus
M
et al. 
Rapamycin-sensitive signalling in long-term consolidation of auditory cortex-dependent memory
.
Eur J Neurosci
 .
2003
;
18
:
942
950
.
61.
Parsons
RG
Gafford
GM
Helmstetter
FJ
.
Translational control via the mammalian target of rapamycin pathway is critical for the formation and stability of long-term fear memory in amygdala neurons
.
J Neurosci
 .
2006
;
26
:
12977
12983
.
62.
Blundell
J
Kouser
M
Powell
CM
.
Systemic inhibition of mammalian target of rapamycin inhibits fear memory reconsolidation
.
Neurobiol Learn Mem
 .
2008
;
90
:
28
35
.
63.
Schicknick
H
Schott
BH
Budinger
E
et al. 
Dopaminergic modulation of auditory cortex-dependent memory consolidation through mTOR
.
Cereb Cortex
 .
2008
;
18
:
2646
2658
.
64.
Banko
JL
Merhav
M
Stern
E
Sonenberg
N
Rosenblum
K
Klann
E
.
Behavioral alterations in mice lacking the translation repressor 4E-BP2
.
Neurobiol Learn Mem
 .
2007
;
87
:
248
256
.
65.
Antion
MD
Merhav
M
Hoeffer
CA
et al. 
Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity
.
Learn Mem
 .
2008
;
15
:
29
38
.
66.
Stoica
L
Zhu
PJ
Huang
W
Zhou
H
Kozma
SC
Costa-Mattioli
M
.
Selective pharmacogenetic inhibition of mammalian target of Rapamycin complex I (mTORC1) blocks long-term synaptic plasticity and memory storage
.
Proc Natl Acad Sci U S A
 .
2011
;
108
:
3791
3796
.
67.
Fortress
AM
Fan
L
Orr
PT
Zhao
Z
Frick
KM
.
Estradiol-induced object recognition memory consolidation is dependent on activation of mTOR signaling in the dorsal hippocampus
.
Learn Mem
 .
2013
;
20
:
147
155
.
68.
Orr
PT
Rubin
AJ
Fan
L
Kent
BA
Frick
KM
.
The progesterone-induced enhancement of object recognition memory consolidation involves activation of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways in the dorsal hippocampus
.
Horm Behav
 .
2012
;
61
:
487
495
.
69.
Toescu
EC
.
Normal brain ageing: models and mechanisms
.
Philos Trans R Soc Lond B Biol Sci
 .
2005
;
360
:
2347
2354
.
70.
Deak
F
Sonntag
WE
.
Aging, synaptic dysfunction, and insulin-like growth factor (IGF)-1
.
J Gerontol A Biol Sci Med Sci
 .
2012
;
67
:
611
625
.
71.
Deli
A
Schipany
K
Rosner
M
et al. 
Blocking mTORC1 activity by rapamycin leads to impairment of spatial memory retrieval but not acquisition in C57BL/6J mice
.
Behav Brain Res
 .
2012
;
229
:
320
324
.
72.
Ma
T
Hoeffer
CA
Capetillo-Zarate
E
et al. 
Dysregulation of the mTOR pathway mediates impairment of synaptic plasticity in a mouse model of Alzheimer’s disease
.
PLoS One
 .
2010
;
5
:
e12845
.
73.
Caccamo
A
Majumder
S
Richardson
A
Strong
R
Oddo
S
.
Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments
.
J Biol Chem
 .
2010
;
285
:
13107
13120
.
74.
Spilman
P
Podlutskaya
N
Hart
MJ
et al. 
Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease
.
PLoS One
 .
2010
;
5
:
e9979
.
75.
Webb
JL
Ravikumar
B
Atkins
J
Skepper
JN
Rubinsztein
DC
.
Alpha-Synuclein is degraded by both autophagy and the proteasome
.
J Biol Chem
 .
2003
;
278
:
25009
25013
.
76.
Pan
T
Rawal
P
Wu
Y
et al. 
Rapamycin protects against rotenone-induced apoptosis through autophagy induction
.
Neuroscience
 .
2009
;
164
:
541
551
.
77.
Majumder
S
Caccamo
A
Medina
DX
et al. 
Lifelong rapamycin administration ameliorates age-dependent cognitive deficits by reducing IL-1β and enhancing NMDA signaling
.
Aging Cell
 .
2012
;
11
:
326
335
.
78.
Halloran
J
Hussong
SA
Burbank
R
et al. 
Chronic inhibition of mammalian target of rapamycin by rapamycin modulates cognitive and non-cognitive components of behavior throughout lifespan in mice
.
Neuroscience
 .
2012
;
223
:
102
113
.
79.
Kim
DH
Sarbassov
DD
Ali
SM
et al. 
mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery
.
Cell
 .
2002
;
110
:
163
175
.

Author notes

Decision Editor: Rafael de Cabo, PhD