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Omega-3 fatty acids (i.e., docosahexaenoic acid; DHA), similar to exercise, improve cognitive function, promote neuroplasticity, and protect against neurological lesion. In this study, we investigated a possible synergistic action between DHA dietary supplementation and voluntary exercise on modulating synaptic plasticity and cognition. Rats received DHA dietary supplementation (1.25% DHA) with or without voluntary exercise for 12 days. We found that the DHA-enriched diet significantly increased spatial learning ability, and these effects were enhanced by exercise. The DHA-enriched diet increased levels of pro-BDNF and mature BDNF, whereas the additional application of exercise boosted the levels of both. Furthermore, the levels of the activated forms of CREB and synapsin I were incremented by the DHA-enriched diet with greater elevation by the concurrent application of exercise. While the DHA diet reduced hippocampal oxidized protein levels, a combination of a DHA diet and exercise resulted in a greater reduction rate. The levels of activated forms of hippocampal Akt and CaMKII were increased by the DHA-enriched diet, and with even greater elevation by a combination of diet and exercise. Akt and CaMKII signaling are crucial step by which BDNF exerts its action on synaptic plasticity and learning and memory. These results indicate that the DHA diet enhance the effects of exercise on cognition and BDNF-related synaptic plasticity, a capacity that may be used to promote mental health and reduce risk of neurological disorders.
The separate applications of voluntary exercise and omega-3 fatty acids dietary supplementation have been shown to affect several parameters of brain plasticity and cognitive function under various experimental conditions in humans and animals (Radak et al., 2001; Churchill et al., 2002; Vaynman et al., 2004; Wu et al., 2004a). The fact that diet and exercise are coexisting components of our daily living suggests that their effects on the brain are complementary. Accordingly, studies were conducted to determine the concurrent action of diet and exercise on molecular systems associated with synaptic plasticity and cognition in the hippocampus.
An increasing body of evidence supports the capacity of exercise to influence brain plasticity and function under homeostatic and challenging conditions. Studies in humans have shown that exercise can decrease cognitive decay associated with aging (Kramer et al., 1999) and is beneficial for reducing the risk of various neurological diseases (Friedland et al., 2001; Laurin et al., 2001). Studies in animals have shown that exercise improves cognitive function during youth and aging stages (Radak et al., 2001; Churchill et al., 2002). The effects of exercise on cognitive function have been linked to the action of brain-derived neurotrophic factor (BDNF) (Vaynman et al., 2004). BDNF is synthesized predominantly by hippocampal neurons, and promotes synaptic facilitation (Kang and Schuman, 1995, Levine et al., 1998, Sherwood and Lo, 1999; Tyler and Pozzo-Miller, 2001). Exercise elevates levels of synapsin I, a nerve terminal phospho-protein involved in neurotransmitter release, axonal elongation and maintenance of synaptic contacts (Wang et al., 1995; Brock and O’Callaghan, 1987), which synthesis (Wang et al., 1995) and phosphorylation (Jovanovic et al., 2000) are affected by BDNF. Cyclic AMP response element binding protein (CREB), a transcription factor involved in learning and memory, is an important modulator of gene expression induced by BDNF (Finkbeiner, 2000). The Akt signaling is important for the effects of a DHA diet on the brain (Akbar et al., 2005) and the effects of BDNF on synaptic plasticity (Yoshii and Constantine-Paton, 2007). CaMKII is a signaling system required for learning and memory (Elgersma et al., 2004) and involved in the effects of exercise on learning and memory performance (Vaynman et al., 2007).
Docosahexaenoic acid (DHA; C22: 6n-3), one of the major omega-3 polyunsaturated fatty acids in the brain, is important for brain development and plasticity, and provides support to learning and memory events in animal models of Alzheimer’s disease (Hashimoto et al., 2002; Lim et al., 2005) and brain injury (Wu et al., 2004a). DHA can affect neural function by enhancing synaptic membrane fluidity and function (Jump, 2002), regulating gene expression (Duplus et al., 2000; Ikemoto et al., 2000; Kitajka et al., 2002; Puskas et al., 2003; Salem et al., 2001), mediating cell signaling (de Urquiza et al., 2000; Jump, 2002; Vaidyanathan et al., 1994), and enhancing long-term potentiation (McGahon et al., 1999).
As discussed above, the effects of the DHA diet and exercise on synaptic plasticity and cognitive function seem to involve similar molecular systems. Accordingly, we have embarked in studies to determine how exercise can integrate its action with diet in order to benefit the CNS. Results indicate that the concurrent combination of exercise and DHA dietary supplementation has greater effects on synaptic plasticity and cognition than their separate applications.
Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA, USA) weighing between 200 and 240g were housed in cages and maintained in environmentally controlled rooms (22–24 °C) with a 12h light/dark cycle. After acclimatization for 1 week on standard rat chow, one set of rats was exposed to a DHA-enriched diet (1.25% DHA), while another set were exposed to regular diet. The rats were then maintained on the diets with or without voluntary exercise for one week. The rats were divided into 4 groups: (1) RD-Sed (regular diet - sedentary), (2) RD-Exc (RD - exercise), (3) DHA-Sed, and (4) DHA-Exc; RD-Sed group was regarded as control. After one week, the rats (n=6 within each group) were tested in the Morris Water maze for learning ability. The rats, still on the diet and exercise, were then killed by decapitation; the fresh tissues including hippocampus were dissected, frozen in dry ice and stored at −70°C until use for biochemical analyses. The diets, fed ad libitum, were provided in powder. The control diet was the standard chow with a ratio of n-6/n-3 at 6:1 (#5001, PMI Nutrition, USA); total fat: 4.5%; arachidonic acid: < 0.01%; Calorie: 4.07kcal/gm. The animals were initially omega-3 sufficient by being maintained on the standard chow, and DHA was supplemented in the control diet. Nordic Naturals, Inc provided this relative pure DHA product. The DHA content was 1.25% in the experimental diet, while the EPA was 0.25%. The DHA content in the diets and n-6/n-3 ratios were in Table 1. All experiments were performed in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal suffering was minimized.
Briefly, the rats were trained in the water maze with 2 consecutive trials per day for 5 days. The rats were placed into the tank facing the wall from one of the equally spaced start locations that were randomly changed every trial. Each trial lasted until the rat found the platform or for a max of 1 min. If the rat failed to find the platform in the allocated time, it was gently placed on the platform. At the end of each trial, the rats were allowed to rest on the platform for 1 min, and the escape latencies were recorded.
Hippocampal tissue was homogenized in a lysis buffer containing 137 mM NaCl, 20 mM Tris–HCl pH 8.0, 1% NP40, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, 0.1 mM benzethonium chloride, 0.5 mM sodium vanadate. The homogenates were then centrifuged; the supernatants collected and total protein concentration was determined according to MicroBCA procedure (Pierce, Rockford, IL), using bovine serum albumin as standard. Levels of pro-BDNF, mature BDNF, phospho-synapsin I (p-syn I), total-synapsin I (t-Syn I), phospho-CREB (p-CREB), total-CREB (t-CREB), phospho-Akt (p-Akt), total Akt (t-Akt), phospho-CaMKII (p-CaMKII), and total-CaMKII (t-CaMKII) were analyzed by western blot. Briefly, protein samples were separated by electrophoresis on an 8% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. Non-specific binding sites were blocked in TBS, overnight at 4°C, with 2% BSA and 0.1% Tween-20. Membranes were rinsed for 10 min in buffer (0.1% Tween-20 in TBS) and then incubated with anti-actin or anti-BDNF, p-syn I, t-Syn I, p-CREB, t-CREB, p-Akt, t-Akt, p-CaMKII, t-CaMKII (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), followed by anti-rabbit IgG horseradish peroxidase-conjugate (Santa Cruz Biotechnology). After rinsing with buffer, the immunocomplexes were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s instructions. The film signals were digitally scanned and then quantified using NIH Image software. Actin was used as an internal control for western blot such that data were standardized according to actin values.
The amounts of oxidized proteins containing carbonyl groups were measured by using an Oxyblot kit (Intergen, Purchase, NY). Briefly, the protein sample (10 μg) was reacted with 1× dinitrophenylhydrazine (DNPH) for 15 min, followed by neutralization with a solution containing glycerol and β-mercaptoethanol. These samples were electrophoresed on an 8% polyacrylamide gel and electrotransferred to a nitrocellulose membrane. After blocking, membranes were incubated overnight with a rabbit anti-DNPH antibody (1:150) at 4°C, followed by incubation in goat anti-rabbit (1:300) for 1 hr at room temperature. After rinsing with buffer, the immunocomplexes were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s instructions. The Oxyblot bands were all grouped together in each group and then analyzed by NIH image software as previous described (Wu, et al., 2004).
Actin was employed as the internal standard for normalizing western blot results. The RD-Sed group was included in each gel to normalize the results of different gels. The RD-Sed group was also used as a control to make comparisons between different groups. The total and activated forms of synapsin I, CREB, Akt, and CaMKII were expressed as a ratio of their phosphorylated forms to their respective total forms. The values were then presented in bar figures and represented the mean ± SEM. The data were analyzed by ANOVA followed by Fisher’s protected least significance post hoc test. Statistical differences were considered significant at P < 0.05.
It has been shown that both DHA dietary supplementation and exercise have beneficial effects on cognitive function. We have embarked in studies to determine a possible synergistic effect of a DHA-enriched diet and exercise on cognition. Rats were exposed to a regular or DHA-enriched diet, with or without exercise for 7 days, followed by a learning test using the Morris Water Maze for five days. The results demonstrated that the DHA-enriched diet-fed rats performed better as evidenced by lower escape latency (21.4±3.4 s, 17.4.4±1.8 s, 13.6±2.5 s) than the rats maintained on the regular diet (34.6±4.5 s, 29.6±3.3 s, 21.7±3.1 s) at days 3,4,5 of cognitive testing (p < 0.05 at all 3 days; Fig. 1A). Furthermore, results showed that exercise boosted the effect of DHA supplementation expressed in much lower escape latency in the exercised rats-fed DHA diet (16.3±4.0 s, 11.3±2.4 s, 8.8±1.1 s) compared to the separate effects of DHA or exercise at days 3,4,5 of cognitive testing (p < 0.05 at all 3 days; Fig. 1A and 1B). The significant difference between these groups at day 5 were shown in Figure 1B.
The processing of mature BDNF (m-BDNF) involves the precursor molecular stage as pro-BDNF, opening the possibility that separate application of diet and exercise can regulate pro-BDNF and m-BDNF. The results showed that pro-BDNF was increased in sedentary rats-fed the DHA diet (138% of RD-Sed, p < 0.05; Fig. 2A) and exercised rats-fed regular diet (128% of RD-Sed, p < 0.05; Fig. 2A). The pro-BDNF levels were much higher in the exercised rats-fed the DHA diet (159% of RD-Sed; p < 0.05) than in sedentary animals-fed DHA diets or exercised rats-fed regular diet (Fig. 2A). The m-BDNF was also higher in the sedentary group-fed DHA diets (125% of RD-Sed; p < 0.05) and the exercised group-fed regular diet (122% of RD-Sed; p < 0.05), which was boosted to 144% (p < 0.05) in the exercised rats-fed DHA diets (Fig. 2B).
BDNF facilitates synaptic transmission through the activation of synapsin I and CREB (Finkbeiner, 2000; Jovanovic et al., 1996; Wang et al., 1995). To evaluate the effects of DHA dietary supplementation and exercise on the molecular systems associated with the action of BDNF on synaptic plasticity, we assessed protein levels of the total and activated forms of synapsin I and CREB in all groups.
The results showed that the ratio of p-synapsin I to total synapsin I was increased in the rats receiving either the DHA-enriched diet (0.62 vs. 0.47; p < 0.05; Fig. 3A) or exercise alone (0.56 vs. 0.47; Fig. 3A) compared to the sedentary rats fed regular diets. Moreover, this ratio was much higher in the group of rats receiving exercise combined with the DHA dietary supplementation (0.89; p < 0.05; Fig. 3A) than those rats receiving either treatment by separate.
The separate applications of DHA dietary enrichment or exercise elevated the ratio of p-CREB to total CREB (0.61 and 0.70 respectively; p<0.05; Fig. 3B) compared to sedentary rats fed regular diets (0.51). Furthermore, much higher ratio of p-CREB to total CREB was found when exercise combined with the DHA-enriched diet (0.90; p < 0.05) compared to the separate effects of the DHA diet or exercise (Fig. 3B).
Recent reports indicate that DHA is a positive modulator of Akt signaling in neuronal survival (Akbar et al., 2005) and that Akt signaling is involved in the effects of BDNF on synaptic plasticity (Yoshii and Constantine-Paton, 2007). The ratio of p-Akt to total Akt was increased in rats supplemented with DHA or receiving exercise alone (0.68 and 0.63 respectively; p < 0.05; Fig. 4A) compared to sedentary rats fed regular diets (0.52). This ratio was much higher in the group of rats that received the combined application of exercise and DHA dietary supplementation (0.87; p < 0.05) compared to the application of each treatment by separate (Fig. 4A). In addition, we found a significant positive correlation between p-Akt and m-BDNF (r = 0.82, p < 0.05; Fig. 4C).
We measured CaMKII levels based on its involvement in the effects of exercise on hippocampal learning and memory (Vaynman et al., 2007). The ratio of p-CaMKII to total CaMKII was increased in rats receiving DHA dietary supplementation or exercise by separate (0.57 and 0.60 respectively; p < 0.05; Fig. 4B) compared to sedentary rats fed regular diets (0.46). The combined application of the DHA diet and exercise resulted in a greater ratio of p-CaMKII to total CaMKII (0.91; p < 0.05; Fig. 4B) compared to separate application of the DHA diet or exercise.
The oxidative stress in rats was assessed using Western blot analysis of DNPH-derivatized carbonyl groups on oxidized proteins in all groups. The results showed that the separated applications of the DHA diet or exercise reduced oxidized protein levels (81% and 78% of RD-Sed; p < 0.05; Fig. 5) compared to their respective control levels. The combined application of the DHA diet and exercise induced greater reduction of oxidized protein levels (64% of RD-Sed, p < 0.05; Fig. 5) compared to the separate applications of the DHA diet or exercise.
Based on reports (King et al., 2006; Huang et al., 2007) suggesting that a tail injection of DHA before dietary DHA supplementation is important for counteracting the effects of spinal cord injury, we performed studies to determine whether a high dose of DHA at the beginning of the experiment might be necessary to detect cognitive enhancement. We injected DHA through tail vein as described by King et al. (2006), followed by DHA diet feeding with or without exercise. Results showed no differences in cognitive performance between the group that received DHA dietary supplementation and the group that received a DHA injection additionally to the dietary DHA supplementation (data not shown).
We have found that a short 12-day exposure to a DHA-enriched diet significantly increases learning ability, and these effects are enhanced by the concurrent application of voluntary exercise. The effects of the DHA diet and exercise on cognitive enhancement were paralleled by elevations in BDNF, and the activated forms of the synaptic proteins CREB, synapsin I, and CaMKII, important for hippocampal learning. Levels of the Akt signaling system were also elevated in proportion to BDNF levels suggesting an action of BDNF on Akt signaling in our paradigm. The enhanced actions of the DHA diet and exercise on cognition and neuroplasticity suggest a possible mechanism by which specific aspects of lifestyle integrate their actions at the molecular level to influence neuronal vitality and function (Fig. 6).
Our results showed that either a DHA-enriched diet or exercise were sufficient to augment spatial learning performance in the Morris water maze. We chose the current exercise paradigm based on our previous studies showing that the same exercise conditions were enough to promote beneficial effects on rat brain function and cognition (Vaynman et al., 2004, 2007). Although DHA dietary supplementation has been shown to improve learning and memory performance, our short-term 12-day exposure protocol clearly differed from previous studies. For example, Lim and Suzuki (2000, 2001) found improved maze learning ability in rats exposed to DHA ethyl ester for more than one month. It has been reported that tail injection of DHA is important for the effects of DHA improving recovery after spinal cord injury in rats (King et al., 2006). However, our studies showed that a high dose of DHA injected at the first day of the treatment did not result in significantly better learning and memory performance, in addition to the one observed after the sole exposure to dietary DHA. The current results imply that a short exposure to DHA dietary supplementation can provide beneficial effects on cognitive function. The current studies demonstrate that the combined applications of the DHA diet and exercise further enhanced spatial learning ability.
Several studies have shown that DHA from dietary supplementation can be incorporated into the brain; however, results across different studies are difficult to compare given the large variety of dietary supplementation protocols involved. Levels of DHA incorporation into the brain are dependent on several factors such as: the degree of DHA deficiency before the treatment, amount of DHA being supplemented, and the length of the supplementation. In our experiments, exercise is an additional factor as it may influence DHA metabolism or incorporation into the brain. Most current methods to assess levels of DHA in the brain are relative to the detection of other C22 fatty acids such as docosatetraeonic acid (DTA). In these terms, it has been reported that a 3-month DHA dietary supplementation (0.6%), produced a 28% elevation of DHA in the brain of mice that had been depleted of DHA (Lim et al., 2005). Based on the above considerations, it is difficult to make any extrapolation to our study in terms of levels of DHA incorporation into the brain. It is important, however, to emphasize that the fact that the effects of our DHA dietary supplementation were not based on prior DHA deficiency provide important information for the design of DHA supplementation programs.
Our current results show that DHA dietary supplementation increases the levels of m-BDNF and pro-BDNF in the hippocampus, and these effects are enhanced by the concurrent application of exercise. It has been known that BDNF is synthesized in the brain as pro-BDNF, which is converted to m-BDNF (Li et al., 2002; Lu, 2003). Both pro-BDNF and m-BDNF have been positively associated with cognitive function and a reduction of both has been linked to cognitive impairment in pre-clinical stages of Alzheimer’s disease (Peng et al., 2005). Another recent report indicates that pro-BDNF is rapidly converted intracellularly to m-BDNF (Matsumoto et al., 2008), suggesting that increased pro-BDNF may contribute to the enhanced elevation of m-BDNF in our paradigm. In the hippocampus, the mature BDNF converted from pro-BDNF at synaptic sites with profound implications for modulation of synaptic plasticity (Lu, 2003). It is known that m-BDNF modulates synaptic plasticity (Kang and Schuman, 1995; Levine et al., 1998; Sherwood and Lo, 1999; Tyler and Pozzo-Miller, 2001; Bolton et al., 2000; Hariri et al., 2003; Thoenen, 1995), and hippocampal m-BDNF is important for cognitive function (Mu et al., 1999) and for induction of long-term potentiation (Patterson et al., 1996; Linnarsson et al., 1997), a physiological correlate of learning. Given the involvement of m-BDNF in the enhancement of learning and memory as a result of exercise (Vaynman et al., 2004), it is possible that m-BDNF can also serve as a mediator for the effects of diet and exercise on cognition in our paradigm.
Several possibilities may explain the effects of our DHA dietary supplementation on the brain. DHA may increase BDNF in the brain using multiple mechanisms: (a) it is known that DHA is converted to Neuroprotectin D1 (NPD1; Lukiw et al. 2005; Mukherjee et al, 2007), which can elevate levels of BDNF; (b) the action of DHA on plasma membranes may activate signaling mechanisms that can result in more BDNF; (c) the antioxidant capacity of DHA may help reduce oxidative stress that has been shown to decrease BDNF (Wu et al., 2004); (d) DHA may help transport glucose across the brain blood barrier to provide energy source for the neurons (Pifferi et al., 2007).
The current results indicate that the DHA dietary supplementation increased the activated state of Akt in the hippocampus, and that this was enhanced by the concurrent application of exercise. In addition, there was a significant positive association between activated state of Akt and mature BDNF. Akt signaling is a crucial step by which BDNF exerts its action on synaptic plasticity, involving NMDA receptor activation (Yoshii and Constantine-Paton, 2007). In addition, the fact that Akt signaling is an important intermediate factor by which DHA impacts neuroplasticity (Akbar et al., 2005) provides special relevance to our results. Overall, the emerging concept is that BDNF activation acting through Akt signaling may play an important role in the beneficial effects of DHA and exercise on synaptic plasticity and cognition (Fig. 6).
The CaMKII is another signaling system which action is critical for learning and memory ability (Elgersma et al., 2004), and plays a role on the effects of exercise on hippocampal-dependent cognitive enhancement (Vaynman et al., 2007). DHA dietary supplementation has been shown to prevent a decrease in CaMKII in a transgenic mouse model of Alzheimer’s disease (Calon et al., 2005). Our findings indicate that exercise and the DHA-enriched diet modulate hippocampal CaMKII activation, and these effects are enhanced by the simultaneous applications of diet and exercise. These results are novel to demonstrate a mechanism by which two aspects of lifestyle can interact at the molecular level with subsequent effects on the modulation of cognitive function.
Exercise has been shown to help cognitive ability using BDNF (Vaynman et al., 2007; Gomez-Pinilla, 2007). Given the impact of the DHA diet and exercise on the BDNF system, we aimed to determine their influence on molecular systems associated with the action of BDNF on synaptic plasticity and cognition. The synthesis (Wang et al., 1995) and phosphorylation (Jovanovic et al., 2000) of synapsin I are affected by BDNF, which can affect neurotransmitter release, axonal elongation and maintenance of synaptic contacts (Wang et al., 1995; Brock and O’Callaghan, 1987). The cyclic AMP response element binding protein (CREB), a transcription factor involved in learning and memory, is an important modulator of gene expression induced by BDNF (Finkbeiner, 2000). We found that DHA increased the activated levels of both synapsin I and CREB in the hippocampus, and that the effects of DHA were enhanced by exercise. Since BDNF and its downstream effectors synapsin I and CREB are involved in learning and memory, our findings suggest that the DHA dietary supplementation and exercise combination may be particularly useful to overcome the vulnerability of the brain to cognitive deterioration (Fig. 6).
Regulation of oxidative stress levels has been related to the effects of diet and exercise on brain energy metabolism (Wu et al., 2004a; Molteni et al., 2004). The present results indicate that the DHA diet reduced levels of protein oxidation and that this effect was enhanced by exercise. These results are important considering that cumulative levels of oxidative stress can damage cell membranes, and that omega-3 fatty acids such as DHA are abundant components of membranes. Recent reports show that anti-oxidant factors such as vitamin E can protect DHA from oxidative damage (Petursdottir et al., 2007). Given that voluntary exercise reduces oxidative stress (Molteni et al., 2004), it is possible that exercise may preserve DHA in the brain, resulting in superior DHA benefits. The potential anti-oxidant action of DHA itself may affect mechanisms that maintain synaptic plasticity, as it has been shown that reduction of oxidative stress can protect against synaptic dysfunction (Wu et al., 2004b). This has been supported by findings that fish oil feeding (a great source of DHA) can provide anti-oxidant effects in animal models of Alzheimer’s disease and aging with subsequent effects on cognition (Hashimoto et al., 2002; Hossain et al., 1999).
The effects of diet and exercise on the brain are receiving increasing recognition, making us to ask what would be the combined effects of both, as it normally occurs under daily living conditions. An unhealthy diet high in saturated fat has been shown to reduce the levels of BDNF-related synaptic plasticity and cognitive function, while the concurrent exposure to exercise compensated for the effects of the diet (Molteni et al., 2004). In turn, the present studies indicate that exercise can boost the benefits of a healthy diet on neuroplasticity. These studies portray BDNF-mediated plasticity as a crucial intermediate mechanism for the effects of diet and lifestyle on the brain. A recent study (Van Praag et at, 2007) has reported that consumption of another dietary component, flavonoids, can also synergize with the ability of exercise to induce neurogenesis in the adult rodent hippocampus. The collaborative effects of diet and exercise are logical extension of our daily routine, which seem to determine the confines of brain plasticity and disease. The present results are significant to suggest that the inherent capacity of the brain to benefit from the effects of DHA dietary supplementation and exercise can be used to overcome neurological disorders affecting cognitive abilities.
This study was supported by NIH award NS50465.
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