Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan
Correspondence to: Satoshi Kida; Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. E-mail: email@example.com
New neurons are generated even during adulthood in mammalian species, including rodents and primates (Altman and Das, 1965; Alvarez-Buylla and Lim, 2004; Eriksson et al., 1998; Zhao et al., 2008). Importantly, adult neurogenesis has been shown to occur in the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus (Doetsch et al., 1997; Seki and Arai, 1993; Seri et al., 2001). Previous studies have also shown that newborn neurons are integrated into the existing dentate gyrus (DG) circuit when they become 2–4 weeks old (Cameron et al., 1993; Kee et al., 2007; van Praag et al., 2002).
The hippocampus is known to play a central role in memory formation such as episodic and spatial memories (Eichenbaum, 2000; Kogan et al., 2000; Scoville and Milner, 1957). Interestingly, recent studies have shown the impact of adult hippocampal neurogenesis on hippocampus-dependent learning and memory. Inhibition of adult hippocampal neurogenesis by X-ray irradiation or pharmacological or genetic manipulation results in the impairment of hippocampus-dependent memory (Arruda-Carvolho et al., 2011; Denny et al., 2012; Ko et al., 2009). In contrast, accelerated adult hippocampal neurogenesis, e.g., via environmental enrichment or exercise, has been suggested to lead to the improvement of hippocampus-dependent memory (Bruel-Jungerman et al., 2005; Nilsson et al., 1999; van Praag et al., 1999, 2000). Furthermore, a recent study has shown that new young neurons (3–8 weeks old) generated through adult neurogenesis are more efficiently recruited into the memory trace and are more plastic compared with older neurons (Gu et al., 2012; Kee et al., 2007; Stone et al., 2011). These findings raise the possibility that these newborn neurons contribute to the improvement of memory performance by efficiently integrating into the memory trace.
Memantine (MEM) is an uncompetitive antagonist of the N-methyl-d-aspartate glutamate receptor (NMDAR; Persons et al., 1999). MEM displays a neuroprotective effect and, thereby, has been used for the treatment of Alzheimer's and Parkinson's disease (Bormann, 1989; Danysz et al., 1997; Müller et al., 1995; Seif el Nasr et al., 1990; Volbracht et al., 2006). Interestingly, a recent study showed that a single intraperitoneal injection of a high dose (50 mg/kg) of MEM more dramatically and transiently promoted adult hippocampal neurogenesis by increasing the proliferation of neural progenitor cells (Maekawa et al., 2009; Namba et al., 2009) compared with the other treatments examined such as exercise and environmental enrichment (Bruel-Jungerman et al., 2005; Nilsson et al., 1999; van Praag et al., 1999, 2000). Importantly, newborn cells generated by treatment with MEM differentiate normally into mature granule neurons (Maekawa et al., 2009). Therefore, treatment with MEM might enable us to examine the age-dependent roles of newborn neurons in the function of the hippocampus, including learning and memory. From these backgrounds, to understand the impact of increased adult hippocampal neurogenesis on memory performance, we examined hippocampus-dependent memory performance in mice with increased hippocampal neurogenesis following treatment with MEM.
MATERIALS AND METHODS
The mice were housed in cages of five or six, maintained on a 12-h light/dark schedule, and allowed ad libitum access to food and water in their home cages. All experiments were conducted during the light phase of the cycle in an illuminated testing room according to the Guide for the Care and Use of Laboratory Animals of the Japan Neuroscience Society and Tokyo University of Agriculture. The animals used in this study were 3-month-old male C57BL/6N mice. All experiments were conducted blind to the treatment condition of the mouse. Animal behavior was recorded using a video camera.
The mice were injected intraperitoneally (i.p.) with MEM (Sigma) at a dose of 50 mg/kg body weight. The control mice were injected with the same volume of 0.9% saline. After 2 days, the mice were injected i.p. with 50 mg/kg body weight of 5-bromo-2-deoxyuridine (BrdU; Sigma) four times at intervals of 2 h.
Before the commencement of the behavioral experiments, the mice were handled individually for 3 min each day for 5 days. We tested at 3 days, 3 or 6 weeks, or 4 months after the injection of MEM using different groups of animals.
Morris water maze task
The Morris water maze task was performed as described previously (Kim et al., 2011; Suzuki et al., 2004). The mice were trained with two trials per day at an interval of 1 min for 6 days (Fig. 1) or 7 days (Fig. 4). The mice were trained at approximately the same time every day. In the probe test at 24 h after training on days 3 and 6 (Fig. 1) or day 7 (Fig. 4), the platform was removed and the mice were allowed to swim for 1 min. We measured the time that the mice spent in each quadrant (opposite [OP], adjacent right [AR], target quadrant [TQ], and adjacent left [AL]).
Social recognition task
The social recognition task was performed as described previously (Fukushima et al., 2008; Nomoto et al., 2012; Suzuki et al., 2011). Adult mice were placed into individual plastic cages, identical to those in which they were normally housed (30 × 17 × 12 cm), in the experimental room. After a period of 60 min, a juvenile mouse was placed into a cage with the adult mouse for 1.5 min (Fig. 2D) or 3 min (Fig. 2B) (first exposure). The duration of the adult's social investigation behavior was quantified using a stopwatch. Social investigation was defined as described previously (Thor and Holloway, 1982). Memory was assessed at 24 h later by recording the length of social investigation time exhibited by the subject to the same juvenile (second exposure) for the same duration as the first exposure (1.5 or 3 min). A recognition index was calculated as the ratio of the social investigation times during the second and first exposures.
After anesthetization, all mice were perfused with 4% paraformaldehyde containing 0.5% picric acid. The brains were then removed, fixed overnight, transferred to 30% sucrose, and stored at −80°C. Coronal sections (14 µm for BrdU/NeuN staining and 30 µm for BrdU/phosphorylated cAMP responsive element binding protein [pCREB] staining) were generated using a cryostat.
For BrdU/NeuN staining (Maekawa et al., 2009), consecutive sections were boiled in citrate buffer solution containing 82 mM sodium citrate and 18 mM citric acid for 5 min and incubated with 2N HCl at 37°C for 30 min, followed by incubation in a blocking solution (TBST buffer plus 10% goat serum albumin). The sections were incubated with a monoclonal rat anti-BrdU primary antibody (1:5000; Novus Biologicals) and a monoclonal mouse anti-NeuN primary antibody (1:500; Millipore) in the blocking solution overnight. Subsequently, the sections were washed with phosphate-buffered saline (PBS) and incubated for 2 h with Alexa Fluor 594 conjugated goat antirat IgG (1:500; Invitrogen) and Alexa Fluor 488 conjugated goat antimouse IgG (1:500; Invitrogen).
For BrdU/pCREB staining (Mamiya et al., 2009), free floating sections were treated with 1% hydrogen peroxide and then incubated overnight with the primary antibody, including rat monoclonal anti-BrdU and rabbit polyclonal anti-pCREB (S133; 1:1500; Millipore). The sections were washed with PBS and then incubated with HRP-conjugated antirabbit secondary antibodies (1:500; Jackson Immunoresearch Laboratories) and incubated in TSA-FITC for 30 min and Alexa Fluor 568-conjugated streptavidin (Invitrogen) for 1 h.
All fluorescence images were acquired using a confocal microscope (FV300; Olympus). Equal cutoff thresholds were applied to all slices using FLUOVIEW software (Olympus).
For BrdU/NeuN staining, BrdU-positive cells throughout the rostrocaudal extent of the DG were counted in every eighth section, and the total number of BrdU-positive cells was calculated by multiplying the count in each section by eight and then totaling the values (Maekawa et al., 2009). BrdU-positive cells were colocalized with NeuN, a marker of mature neurons, and were counted at 4 weeks and 4 months after treatment with MEM, whereas all BrdU-positive cells were counted at 1 week after treatment with MEM.
For BrdU/pCREB staining, pCREB-positive cells in the DG region of the hippocampus (Bregma −1.70, −2.80, and −3.80 mm) were quantified using a computerized image analysis system, as described previously (Winroof version 5.6 software; Mitani Corporation) (Frankland et al., 2006; Mamiya et al., 2009; Zhang et al., 2011), and confocal 2-μm z-stack images were obtained using FLUOVIEW software to detect BrdU and pCREB double-positive cells.
Data were analyzed using analysis of the variance (ANOVA). One-way or two-way ANOVA followed by a post hoc Newman–Keuls comparison were used to analyze the effects of groups, time, and drugs. A paired t test was used to analyze differences of the social investigation times within each group between the first and second exposures in the social recognition task and to analyze differences of the time spent in TQ compared with the other quadrants in the Morris water maze task. The Pearson's correlation test was used to analyze the relationship between the number of BrdU-positive cells and the time spent in TQ. All values in the text and figure legends represent the mean ± standard error of the mean.
Hippocampus-Dependent Memory Enhancement at 3 Weeks After MEM Treatment
To examine the effects of treatment with MEM on memory performance, the mice were subjected to two types of hippocampus-dependent tasks. We first performed the Morris water maze task to assess spatial memory performance, at 3 days, 3, 6 weeks or 4 months after the systemic injection of MEM. Training took place over 6 days, during which the mice were given two trials to find a hidden platform. Two-way ANOVA comparing the escape latencies during training revealed no effect of drug and time × drug interaction (Fig. 1A,D,G,J; 3 days, time, F(5,33) = 45.732, P < 0.05; drug, F(1,33) = 0.042, P > 0.05; time × drug interaction, F(5,33) = 0.235, P > 0.05; 3 weeks, time, F(5,28) = 28.895, P < 0.05; drug, F(1,28) = 0.043, P > 0.05; time × drug interaction, F(5,28) = 0.9104, P > 0.05; 6 weeks, time, F(5,17) = 20.507, P < 0.05; drug, F(1,17) = 0.139, P > 0.05; time × drug interaction, F(5,17) = 0.488, P > 0.05; 4 months, time, F(5,41) = 37.748, P < 0.05; drug, F(1,41) = 0.137, P > 0.05; time × drug interaction, F(5,41) = 0.645, P > 0.05), indicating that all groups displayed comparable escape latencies. To measure the formation of spatial memory, the mice were given a probe trial in which the platform was removed from the pool at 24 h after training on day 3 (probe test 1). Only the group trained at 3 and 6 weeks after treatment with MEM searched selectively in TQ compared with the other quadrants during the probe trial, while the other MEM- and saline-treated groups failed to do so (Fig. 1B,E,H,K; 3 and 6 weeks after treatment with MEM, TQ vs. OP, AR, and AL, paired t test, Ps < 0.05; the other groups, paired t test, Ps > 0.05), indicating that this group formed a spatial memory. These observations suggest that spatial memory was improved when the mice were trained at 3 or 6 weeks, but not at 3 days or 4 months, after treatment with MEM.
We further performed a second probe test (probe test 2) at 24 h after training on day 6. In contrast to the results from probe test 1, all groups searched selectively in TQ compared with the other quadrants (Fig. 1C,F,I,L; TQ vs. OP, AR and AL; paired t test, Ps < 0.05), indicating that they formed a spatial memory. However, the mice trained at 3 or 6 weeks after treatment with MEM spent a comparable time in TQ as the other groups, indicating that this group displayed normal spatial memory. As a previous study showed that transgenic mice expressing a dominant active mutant of CREB displayed improved memory performance even after extensive training (for 6 days) (Suzuki et al., 2011), our observations indicated that the memory enhancement observed following treatment with MEM was significant, but mild (compared with other treatments, such as the upregulation of CREB activity).
We next performed the social recognition task at 3 days, 3 weeks, or 4 months after MEM treatment. MEM- or saline-treated male mice were exposed to a juvenile male mouse twice for 3 min at an interval of 24 h, and we assessed the difference in time taken to investigate the juvenile between the first and second exposures. Two-way ANOVA comparing recognition indices revealed no significant effect of time, drug, and time × drug interaction (Fig. 2A; time, F(2,67) = 1.070, P > 0.05; drug, F(1,67) = 0.092, P > 0.05; time × drug interaction, F(2,67) = 0.011, P > 0.05). Consistently, all groups showed significant decreases in social investigation time at the second exposure compared with the first exposure (Fig. 2B; Ps < 0.05), suggesting that they formed a social recognition memory. Similarly with the observations in the Morris water maze task, it is possible that a weaker training protocol could facilitate the observation of memory improvement following treatment with MEM. To examine this, we performed the social recognition task using a weaker training protocol (1.5 min exposure) (Suzuki et al., 2011). Two-way ANOVA revealed a significant time × drug interaction (Fig. 2C; time, F(2,121) = 1.732, P > 0.05; drug, F(1,121) = 1.803, P > 0.05; time × drug interaction, F(2,121) = 4.577, P < 0.05). The post hoc Newman–Keuls test showed that the mice treated with MEM at 3 weeks before training displayed a significantly better recognition index than the other groups (Fig. 2C; Ps < 0.05). Consistently, only the group treated with MEM at 3 weeks before training displayed a significant decrease in investigation time at the second exposure (Fig. 2D; paired t test, P < 0.05). These observations are consistent with the results shown in Fig. 1 and suggested that treatment with MEM leads to a time-dependent improvement of hippocampus-dependent memory performance at approximately 3 weeks after treatment.
Positive Correlation Between the Number of BrdU-Positive Cells and Spatial Memory Performance in MEM-Treated Mice
We next examined the relationship between enhanced neurogenesis following treatment with MEM and improved memory performance. We first measured the number of BrdU-positive cells in the DG following the behavioral experiments at 24 h after probe test 2 in the Morris water maze task (Fig. 1). Consistent with a previous study (Maekawa et al., 2009), the MEM-treated groups showed significantly more BrdU-positive cells than the control groups at each time point (Fig. 3A,B; t test, Ps < 0.05), indicating that treatment with MEM promoted adult hippocampal neurogenesis. We next performed correlational analysis using the mice that were subjected to the Morris water maze task at 3 weeks after treatment with MEM by comparing the number of BrdU-positive cells with the memory score (time spent in TQ) in each mouse at the probe test 1. Interestingly, MEM-treated, but not saline-treated, mice demonstrated a significant positive correlation between the number of BrdU-positive cells and the time spent in TQ (Fig. 3C,D; saline, r = −0.53, P > 0.05; MEM, r = 0.77, P < 0.05), suggesting that MEM-treated mice showing more neurogenesis formed a stronger spatial memory. Thus, our observation suggests that increased neurogenesis contributed to the enhancement of hippocampus-dependent memory performance.
New Neurons Generated by Treatment with MEM are Integrated into the Memory Trace
Younger neurons (3–8 weeks old) generated by adult hippocampal neurogenesis have been shown to be selectively integrated into the memory trace (Cameron et al., 1993; Kee et al., 2007). We asked whether the newborn neurons generated by treatment with MEM are also integrated into the hippocampal memory trace. CREB is thought to be activated through its phosphorylation at S133 in neurons participating in the memory trace when the memory is retrieved (Han et al., 2007, 2009). Therefore, we compared the number of BrdU- and/or pCREB-positive cells following the retrieval of a spatial memory. The mice were treated with MEM and BrdU as described above. After 3 weeks of these treatments, the mice were trained with two trials per day for 7 days to allow the formation of a comparable spatial memory in the saline- and MEM-treated groups (Morris water maze [MWM] group) (Fig. 4A). Consistent with the results shown in Fig. 1, both groups formed a spatial memory (Fig. 4B; paired t test, TQ vs. OP, AR, and AL, Ps < 0.05) and spent a comparable time in TQ during the probe trial at 24 h after day 7 (t test, P > 0.05). BrdU- and/or pCREB-positive cells were analyzed at 60 min after the probe trial. The control groups were treated similarly except that the mice were not subjected to the Morris water maze task but stayed in their home cage throughout the experiments (HC group). Consistent with our previous observations, two-way ANOVA comparing BrdU-positive cells between the groups revealed a significant effect of drug and no significant effect of MWM or MWM × drug interaction (Fig. 4C,D; MWM, F(1,36) = 0.045, P > 0.05; drug, F(1,36) = 40.591, P < 0.05; MWM × drug interaction, F(1,36) = 0.020, P > 0.05). On the other hand, two-way ANOVA comparing pCREB-positive cells between the groups revealed a significant effect of MWM and no significant effect of drug or MWM × drug interaction (Fig. 4C,E; MWM, F(1,36) = 11.912, P < 0.05; drug, F(1,36) = 0.015, P > 0.05; MWM × drug interaction, F(1,36) = 0.027, P > 0.05), suggesting that the MWM groups activated spatial memory traces. Importantly, there was no significant difference in the number of pCREB-positive cells between the MEM- and saline-treated groups, suggesting that the MWM groups showed similar spatial memory traces. To clarify the integration of BrdU-positive newborn neurons into the memory trace, we calculated the ratio of double-positive cells (pCREB+/BrdU+) and pCREB-single-positive cells in the MWM groups. The MEM-treated group displayed a significantly higher proportion of double-positive cells compared with the saline-treated group (Fig. 4F; t test, P < 0.05). Importantly, the ratio of the number of double-positive cells between the MEM- and saline-treated groups was comparable with that of the number of BrdU-positive cells between the MEM- and saline-treated groups (Fig. 4G; t test, P > 0.05). These observations suggest that the number of younger neurons integrated into the memory trace reflects the increase in adult hippocampus neurogenesis induced by treatment with MEM. Taken together, our results suggest that the new neurons generated by treatment with MEM were integrated into memory trace similarly to new neurons generated endogenously by adult neurogenesis and contribute to the formation of a memory circuit.
A single treatment of mice with a high dose of MEM has been shown to increase adult hippocampal neurogenesis dramatically (Maekawa et al., 2009). Recent studies have suggested that adult hippocampal neurogenesis plays modulatory roles in hippocampus-dependent memory performance (Arruda-Carvolho et al., 2011; Denny et al., 2012; Ko et al., 2009). In this study, we examined the effects of treatment with MEM on hippocampus-dependent memory and tried to understand the impact of increased hippocampal neurogenesis on memory performance. Our results indicated that treatment with MEM enhanced two types of hippocampus-dependent memories when the mice were trained and tested at 3–6 weeks, but not sooner or later, after treatment with MEM. As the majority of newborn neurons generated through adult neurogenesis has been shown to be integrated into the existing DG circuit at the age of 2–4 weeks (Hastings et al., 1999; Tashiro et al., 2006; van Praag et al., 2002; Zhao et al., 2006), our observations suggest that mice treated with MEM displayed improved memory performance when the newborn neurons that were increased by treatment with MEM became young neurons (approximately 3 weeks old). Additionally, we found that 3-week-old young neurons generate by treatment with MEM were incorporated into the trace of spatial memory similarly to neurons of the same age that were generated through endogenous neurogenesis. Most importantly, a positive correlation was observed between the number of 3-week-old neurons (BrdU-positive cells) and memory scores (time spent in TQ in the probe test 1) in MEM-treated, but not saline-treated, mice. This positive correlation was not observed in both MEM- and saline-treated mice in the probe test 2 (data not shown). Similarly, previous studies have shown no correlation between adult hippocampal neurogenesis and time in TQ in wild-type (WT) mice (Kempermann et al., 2002; Merrill et al., 2003; Snyder et al., 2005). Therefore, these observations strongly suggest that the young neurons generated by treatment with MEM greatly contributed to the improvement of spatial memory. Taken together, our results suggest that treatment with MEM temporally improves hippocampus-dependent memory by increasing adult neurogenesis.
Interestingly, previous studies have shown that young neurons (3–8 weeks old) generated through neurogenesis are incorporated more frequently into the memory trace and are more plastic compared with the other generations of neurons (Gu et al., 2012; Kee et al., 2007; Stone et al., 2011). Taken together with this, our observation that 3-week-old neurons might contribute to the improvement of memory performance raises the possibility that the number of neurons with this age is a critical parameter to determine learning and memory ability, i.e., increased numbers of 3-week-old neurons enhance hippocampus-dependent memory.
Treatment with MEM has been known to promote adult neurogenesis dramatically in the hippocampus compared to other treatments such as exercise and environmental enrichment (Bruel-Jungerman et al., 2005; Nilsson et al., 1999; van Praag et al., 1999, 2000). However, it has remained unknown whether newborn neurons generated by treatment with MEM show a comparable function with those generated by endogenous neurogenesis. We found that MEM-generated newborn neurons were incorporated into the memory trace similarly to naturally generated new neurons, suggesting that the MEM-generated neurons display a normal function. In addition, our results suggest that treatment with MEM is a useful technique to clarify the age-dependent role of newborn neurons in hippocampal function, including learning and memory.
MEM has been used for the treatment of Alzheimer's disease (AD) since MEM shows a neuroprotective effect. Importantly, a previous study has shown that low dose clinically used of MEM (7.5 mg/kg body weight daily for 2 weeks) is enough to increase in neurogenesis in mice (Jin et al., 2006). Taken together with our observations, the possibility is raised that therapeutic effects of MEM in AD might be mediated in part through increase in adult neurogenesis.
MEM is an antagonist of NMDAR (Persons et al., 1999); it is thought to not only function as a strong inducer of adult neurogenesis, but also affects other biological phenomena through the inactivation of NMDAR (De Felica et al., 2007; Marvanová et al., 2001). Therefore, we could not exclude the possibility that MEM improves memory performance through additional mechanisms and not only through increased neurogenesis. Nevertheless, our observations that memory enhancement was not observed 3 days after MEM treatment and that MEM-treated mice exhibited a positive correlation between memory performance and adult neurogenesis strongly suggest that MEM treatment improves memory performance through increase in adult hippocampal neurogenesis. Further studies are required to clarify the roles of new neurons generated by treatment with MEM in hippocampus-dependent memory by examining the effects of specifically blocking adult neurogenesis following treatment with MEM on memory performance.
In this study, young neurons are suggested to contribute to the improvement of hippocampus-dependent memory. Importantly, recent studies have shown that neurons in the amygdala with higher CREB activity are recruited more frequently into the trace of a cued fear memory (Hen et al., 2007, 2009; Zhou et al., 2009). Furthermore, the genetic upregulation of CREB activity in the forebrain has been shown to improve memory formation (Suzuki et al., 2011). Therefore, it is possible that young neurons show higher CREB activity; thereby, playing a positive role in memory formation. Thus, it is important to measure the activity of CREB in 3-week-old young neurons.
However, it is important to note that the improvement of memory observed in the MEM-treated mice was much milder than in transgenic mice expressing a dominant active mutant of CREB that displayed improved memory performance even after extensive training (Suzuki et al., 2011). Therefore, further investigations are required to understand the molecular mechanisms by which MEM-generated young adult neurons improve memory performance in comparison with those following the upregulation of CREB activity.
In summary, mice treated with MEM displayed improved hippocampus-dependent memory when they were trained at 3–6 weeks, but not at 3 days or 4 months, after treatment. As newborn neurons are known to be integrated into the existing DG circuit at the age of 2–4 weeks (Cameron et al., 1993; Kee et al., 2007) and the memory scores were positively correlated with the number of 3-week-old young neurons, we concluded that the new neurons generated by treatment with MEM contributed to the improvement of hippocampus-dependent memory when they become young neurons. Our findings raise the possibility that MEM functions as a memory enhancer that could be used to improve cognitive impairment such as deficits in learning and memory.