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The abilities of docosahexaenoic acid (DHA) and exercise to counteract cognitive decay after traumatic brain injury (TBI) is getting increasing recognition; however, the possibility that these actions can be complementary remains just as an intriguing possibility. Here we have examined the likelihood that the combination of diet and exercise has the added potential to facilitate functional recovery following TBI. Rats received mild fluid percussion injury (mFPI) or sham injury and then were maintained on a diet high in DHA (1.2% DHA) with or without voluntary exercise for 12days. We found that FPI reduced DHA content in the brain, which was accompanied by increased levels of lipid peroxidation assessed using 4-hydroxy-2-hexenal (4-HHE). FPI reduced the enzymes acyl-CoA oxidase 1 (Acox1) and 17β-hydroxysteroid dehydrogenase type 4 (17β-HSD4), and the calcium-independent phospholipases A2 (iPLA2), which are involved in metabolism of membrane phospholipids. FPI reduced levels of syntaxin-3 (STX-3), involved in the action of membrane DHA on synaptic membrane expansion, and also reduced brain-derived neurotrophic factor (BDNF) signaling through its tyrosine kinase B (TrkB) receptor. These effects of FPI were optimally counteracted by the combination of DHA and exercise. Our results support the possibility that the complementary action of exercise is exerted on restoring membrane homeostasis after TBI, which is necessary for supporting synaptic plasticity and cognition. It is our contention that strategies that take advantage of the combined applications of diet and exercise may have additional effects to the injured brain.

Exercise has been shown to impact brain plasticity and function by involving the action of brain-derived neurotrophic factor (BDNF); however, mechanisms involved are poorly understood. Two types of BDNF coexist in the brain, the precursor (proBDNF) and its mature product (mBDNF), which preferentially bind specific receptors and exert distinct functions. It is crucial to understand how exercise affects crucial steps in the BDNF processing and signaling to evaluate therapeutic applications. We found that 7 days of voluntary exercise increased both pro and mature BDNF in the rat hippocampus. Exercise also increased the activity of tissue-type plasminogen activator (tPA), a serine proteinase shown to facilitate proBDNF cleavage into mBDNF. The blockade of tPA activity reduced the exercise effects on proBDNF and mBDNF. The tPA blocking also inhibited the activation of TrkB receptor, and the TrkB signaling downstream effectors phospho-ERK, phospho-Akt, and phospho-CaMKII. The blocking of tPA also counteracted the effects of exercise on the plasticity markers phospho-synapsin I and growth-associated protein 43 (GAP-43). These results indicate that the effects of exercise on hippocampal plasticity are dependent on BDNF processing and subsequent TrkB signaling, with important implications for neuronal function.

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-brain-derived neurotrophic factor (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 enhanced 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.

We have previously shown that the spinal cord is capable of learning a sensorimotor task in the absence of supraspinal input. Given the action of brain-derived neurotrophic factor (BDNF) on hippocampal learning, the current studies examined the role of BDNF in spinal learning. BDNF is a strong synaptic facilitator and, in association with other molecular signals (e.g. cAMP-response element binding protein (CREB), calcium/calmodulin activated protein kinase II (CaMKII) and synapsin I), important for learning. Spinally transected rats given shock to one hind leg when the leg extended beyond a selected threshold exhibited a progressive increase in flexion duration that minimized shock exposure, a simple form of instrumental learning. Instrumental learning resulted in elevated mRNA levels of BDNF, CaMKII, CREB, and synapsin I in the lumbar spinal cord region. The increases in BDNF, CREB, and CaMKII were proportional to the learning performance. Prior work has shown that instrumental training facilitates learning when subjects are tested on the contralateral leg with a higher response criterion. Pretreatment with the BDNF inhibitor TrkB-IgG blocked this facilitatory effect, as did the CaMKII inhibitor AIP. Intrathecal administration of BDNF facilitated learning when subjects were tested with a high response criterion. The findings indicate that instrumental training enables learning and elevates BDNF mRNA levels within the lumbar spinal cord. BDNF is both necessary, and sufficient, to produce the enabling effect.

We found that a single week of exercise enhanced cognitive function on the Morris water maze (MWM), such that exercise animals were significantly better than sedentary controls at learning and recalling the location of the platform. In order to elucidate the role that calcium calmodulin protein kinase II (CAMKII) holds in mediating the exercise-induced enhancement in learning and memory, a specific antagonist of CAMKII, KN-62, was used to block CAMKII in the rat hippocampus during a 1-week voluntary exercise period. Following, a two-trial-per-day MWM was performed for five consecutive days, succeeded by a probe trial 2 days later. Inhibiting CAMKII action during exercise blocked the ability of exercise to enhance memory retention on the MWM; the recall abilities of exercise animals receiving the CAMKII blocker were significantly worse than those of both sedentary and exercise controls. Conversely, CAMKII may not play a significant role in mediating the effects of exercise on learning acquisition as inhibiting CAMKII failed to block the exercise-induced enhancement in learning acquisition. Our results also show that CAMKII activation early during MWM learning may be counterproductive to learning acquisition, as exercising animals given the CAMKII inhibitor performed significantly (P

Voluntary exercise leads to an upregulation of brain-derived neurotrophic factor (BDNF) and associated proteins involved in synaptic function. Activity-induced enhancement of neuroplasticity may be considered for the treatment of traumatic brain injury (TBI). Given that during the first postinjury week the brain is undergoing dynamic restorative processes and energetic changes that may influence the outcome of exercise, we evaluated the effects of acute and delayed exercise following experimental TBI. Male Sprague-Dawley rats underwent either sham or lateral fluid-percussion injury (FPI) and were housed with or without access to a running wheel (RW) from postinjury days 0-6 (acute) or 14-20 (delayed). FPI alone resulted in significantly elevated levels of hippocampal phosphorylated synapsin I and phosphorylated cyclic AMP response element-binding-protein (CREB) at postinjury day 7, of which phosphorylated CREB remained elevated at postinjury day 21. Sham and delayed FPI-RW rats showed increased levels of BDNF, following exercise. Exercise also increased phosphorylated synapsin I and CREB in sham rats. In contrast to shams, the acutely exercised FPI rats failed to show activity-dependent BDNF upregulation and had significant decreases of phosphorylated synapsin I and total CREB. Additional rats were cognitively assessed (learning acquisition and memory) by utilizing the Morris water maze after acute or delayed RW exposure. Shams and delayed FPI-RW animals benefited from exercise, as indicated by a significant decrease in the number of trials to criterion (ability to locate the platform in 7 s or less for four consecutive trials), compared with the delayed FPI-sedentary rats. In contrast, cognitive performance in the acute FPI-RW rats was significantly impaired compared with all the other groups. These results suggest that voluntary exercise can endogenously upregulate BDNF and enhance recovery when it is delayed after TBI. However, when exercise is administered to soon after TBI, the molecular response to exercise is disrupted and recovery may be delayed.

We have conducted studies to determine the potential of dietary factors to affect the capacity of the brain to compensate for insult. Rats were fed with a high-fat sucrose (HFS) diet, a popularly consumed diet in industrialized western societies, for 4 weeks before a mild fluid percussion injury (FPI) or sham surgery was performed. FPI impaired spatial learning capacity in the Morris water maze, and these effects were aggravated by previous exposure of the rats to the action of the HFS diet. Learning performance decreased according to levels of brain-derived neurotrophic factor (BDNF) in individual rats, such that rats with the worst learning efficacy showed the lowest levels of BDNF in the hippocampus. BDNF immunohistochemistry localized the decreases in BDNF to the CA3 and dentate gyrus of the hippocampal formation. BDNF has a strong effect on synaptic plasticity via the action of synapsin I and cAMP-response element-binding protein (CREB), therefore, we assessed changes in synapsin I and CREB in conjunction with BDNF. Levels of synapsin I and CREB decreased in relation to decreases in BDNF levels. The combination of FPI and the HFS diet had more dramatic effects on the active state (phosphorylated) of synapsin I and CREB. There were no signs of neurodegeneration in the hippocampus of any rat group assessed with Fluoro-Jade B staining. The results suggest that FPI and diet impose a risk factor to the molecular machinery in charge of maintaining neuronal function under homeostatic and challenging situations.

Emerging evidence indicates that the expression of trophic factors in the brain is regulated in an activity-dependent manner, which suggests an involvement of trophic factors in events controlled by input activity. We have investigated the possibility that visual sensory input impacts the expression of basic fibroblast growth factor and its receptor in the brain. Rats were maintained for seven days in darkness and then re-exposed to normal illumination for 0, 1, 3 or 6 h. We assessed relative levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs using nuclease protection assays, and examined possible changes in the phenotypic expression of basic fibroblast growth factor and its receptor using immunohistochemistry. There was a significant decrease in levels of basic fibroblast growth factor and fibroblast growth factor receptor messenger RNAs as a result of dark rearing, and levels of messenger RNAs increased progressively with light re-exposure. Changes in messenger RNAs were observed primarily in the cerebral cortex (caudal portion) and were accompanied by alterations in the staining intensity and density of cells exhibiting basic fibroblast growth factor and fibroblast growth factor receptor phenotypes. Regulation of the basic fibroblast growth factor system by sensory input suggests that basic fibroblast growth factor, and perhaps other trophic factors, are mediators of the effects of experience on the structure and function of the CNS.

New evidence indicates that neural activity regulates the expression of trophic factors in the brain but regulation of these molecules by select aspects of behaviour remains solely a fascinating possibility. We report that following training in the Morris water maze, a spatial memory task, the hippocampus and cerebellum of learning rats exhibited an increase in basic fibroblast growth factor messenger RNA. Basic fibroblast growth factor messenger RNA levels were higher during the learning of the task and decreased once asymptotic performance was reached, suggesting an involvement of basic fibroblast growth factor in learning/memory. An active control group, which exercised for the same time as the learning group but the spatial learning component of the task was minimized, exhibited a minor increase in basic fibroblast growth factor messenger RNA. The intensification of the physical activity component of the task by massed or intensive training resulted in greater increases in basic fibroblast growth factor messenger RNA for both learning and yoked groups, but levels of basic fibroblast growth factor messenger RNA in the learning group remained higher than yoked only in the cerebellum. Changes in basic fibroblast growth factor were accompanied by an increase in astrocyte density in the hippocampus in agreement with described roles of basic fibroblast growth factor in astrocyte proliferation/reactivity. Results suggest that learning potentiates the effects of physical activity on trophic factor induction in select brain regions. Trophic factor involvement in behaviour may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to improve rehabilitation and successful ageing.

Increasing evidence indicates that heparan sulfate proteoglycans have a critical role in the regulation of the activity of basic fibroblast growth factor by interacting with it or its receptor. In this study we examined the possibility that heparan sulfate can modulate the basic fibroblast growth factor system at a more fundamental level than activity regulation, by influencing the synthesis of basic fibroblast growth factor and its receptor messenger RNAs. Previous studies in vitro indicate that basic fibroblast growth factor promotes proliferation and differentiation of astrocytes. Accordingly, we examined the possibility that the action of heparan sulfate on the basic fibroblast growth factor system could have a critical role in the modulation of reactivity and/or proliferation of astrocytes in vitro and in vivo . We report that basic fibroblast growth factor applied to pure astrocyte cultures or rat neocortex promoted an increase in the messenger RNA for basic fibroblast growth factor itself and for its receptor. Furthermore, basic fibroblast growth factor applied directly into the brain elicited an increase in messenger RNA for the astrocytic marker glial fibrillary acidic protein. All of these actions, both in vitro and in vivo , were highly potentiated when heparan sulfate was applied in combination with basic fibroblast growth factor. These results suggest that basic fibroblast growth factor regulates astrocytic proliferation or reactivity via an autocrine cascade that involves induction of its own receptor, and that this action is modulated by heparan sulfate.

Basic fibroblast growth factor is a potent trophic factor with a wide spectrum of activity at various stages of neuronal development. In our studies on the effects of select lesions on the expression of growth factors, we observed that neurons of layer VIb of the rat cerebral cortex developed immunoreactivity for basic fibroblast growth factor and its receptor following injury. Recent evidence indicates that layer VIb of the rat cerebral cortex contains the subplate cell population, a group of neurons shown to participate in the development of the cerebral cortex. In this article, we examined the nature and time-course of the response to injury of the expression of basic fibroblast growth factor and its receptor in these cells. We used an anti-basic fibroblast growth factor monoclonal antibody that recognizes the active form of basic fibroblast growth factor, and a polyclonal antibody that recognizes the extracellular domain of the basic fibroblast growth factor receptor. The induction of basic fibroblast growth factor and its receptor in layer VIb cells occurred after entorhinal cortex lesion, fimbria-formix transection or aspiration of small segment of the frontoparietal cortex. The lesion-induced effect was transient, appearing by postlesion day 2 and having disappeared by postlesion day 7. These findings suggest that endogenous basic fibroblast growth factor may have a neuroprotective role on layer VIb neurons after trauma and/or may participate in cortical plasticity during adulthood.

Traumatic brain injury (TBI) is followed by an energy crisis that compromises the capacity of the brain to cope with challenges, and often reduces cognitive ability. New research indicates that events that regulate energy homeostasis crucially impact synaptic function and this can compromise the capacity of the brain to respond to challenges during the acute and chronic phases of TBI. The goal of the present study is to determine the influence of the phenolic yellow curry pigment curcumin on molecular systems involved with the monitoring, balance, and transduction of cellular energy, in the hippocampus of animals exposed to mild fluid percussion injury (FPI). Young adult rats were exposed to a regular diet (RD) without or with 500 ppm curcumin (Cur) for four weeks, before an FPI was performed. The rats were assigned to four groups: RD/Sham, Cur/Sham, RD/FPI, and Cur/FPI. We found that FPI decreased the levels of AMP-activated protein kinase (AMPK), ubiquitous mitochondrial creatine kinase (uMtCK) and cytochrome c oxidase II (COX-II) in RD/FPI rats as compared to the RD/sham rats. The curcumin diet counteracted the effects of FPI and elevated the levels of AMPK, uMtCK, COX-II in Cur/FPI rats as compared to RD/sham rats. In addition, in the Cur/sham rats, AMPK and uMtCK increased compared to the RD/sham. Results show the potential of curcumin to regulate molecules involved in energy homeostasis following TBI. These studies may foster a new line of therapeutic treatments for TBI patients by endogenous upregulation of molecules important for functional recovery.

Clinical evidence indicates that motor training facilitates functional recovery after a spinal cord injury (SCI). Brain-derived neurotrophic factor (BDNF) is a powerful synaptic facilitator and likely plays a key role in motor and sensory functions. Spinal cord hemisection decreases the levels of BDNF below the injury site, and exercise can counteract this decrease [Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F (2005) Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp Neurol 193:411-419]. It is not clear, however, whether the exercise-induced increases in BDNF play a role in mediating the recovery of locomotion after a SCI. We performed a lateral cervical ( approximately C4) hemisection in adult rats. Seven days after hemisection, the BDNF inhibitor trkB IgG was injected into the cervical spinal cord below the lesion ( approximately C5-C6). Half of the rats were exposed to voluntary running wheels for 14 days. Locomotor ability was assessed by determining the symmetry between the contralateral (unaffected) vs. the ipsilateral (affected) forelimb at the most optimum treadmill speed for each rat. Sedentary and exercised rats with BDNF inhibition showed a higher level of asymmetry during the treadmill locomotion test than rats not treated with the BDNF inhibitor. In hemisected rats, exercise normalized the levels of molecules important for synaptic function, such as cyclic AMP response element binding protein (CREB) and synapsin I, in the ipsilateral cervical enlargement, whereas the BDNF blocker lessened these exercise-associated effects. The results indicate that BDNF levels play an important role in shaping the synaptic plasticity and in defining the level of recovery of locomotor performance after a SCI.

Traumatic brain injury (TBI) continues to be one of the leading causes of mortality and morbidity, with approximately 5.3 million Americans suffering from enduring disabilities resulting from TBI (Binder et al., 2005). Despite many experimental and clinical efforts using numerous therapeutic approaches, there are currently no proven treatments to alleviate the sequelae of TBI or to enhance recovery of function. Our laboratories have been investigating treatment strategies that may facilitate endogenous repair mechanisms and enhance functional recovery after experimental TBI in the rat. One approach has been to use spontaneous or voluntary exercise, obtained via running wheel (RW) exposure. Exercise is known to increase key molecules involved in neuroplasticity, such as brain-derived neurotrophic factor (BDNF) and synapsin I (Neeper et al., 1995; van Praag et al., 1999; Cotman and Berchtold, 2002), as well as reduce markers of oxidative stress (Navarro et al., 2004; Wu et al., 2004; Pan et al., 2007). While experimental TBI can acutely decrease expression of BDNF mRNA within the hippocampus (Hellmich et al., 2005), most studies have reported hippocampal increases in BDNF mRNA within the first 24 h following TBI (Oyesiku et al., 1999; Truettner et al., 1999; Morrison et al., 2000). At later post-injury times, neither hippocampal BDNF mRNA (Hicks et al., 2002) nor BDNF protein differs from that of controls (Griesbach et al., 2004b, 2007; Chen et al., 2005). However, spontaneous exercise in rats with TBI can increase the expression and/or levels of BDNF and synapsin I within the hippocampus (Hicks et al., 1998; Griesbach et al., 2004b, 2007), and this effect is associated with an improvement in cognitive performance (Griesbach et al., 2004b). The timing of this post-injury exercise has been shown to play an important role after experimental fluid percussion injury (FPI) to the brain. We previously reported that voluntary exercise during the first week after sham injury increased hippocampal BDNF and synapsin I protein, but exercise did not increase these molecules after a FPI (Griesbach et al., 2004a, 2007). The mechanisms underlying the inability of acute exercise to up-regulate BDNF after TBI are not known, but may be related to alterations in neurotransmitter systems during this acute period following injury. Transmitter depletion studies have shown that norepinephrine (NE) is necessary for voluntary wheel-running to up-regulate BDNF mRNA in the hippocampus (Garcia et al., 2003). Anti-depressant drugs, which increase synaptic levels of NE and 5-HT, have been reported to increase levels of hippocampal BDNF mRNA (Nibuya et al., 1995) and protein (Peng et al., 2008; Kozisek et al., 2008). In addition, anti-depressant drugs strengthen BDNF mRNA up-regulation when given in combination with exercise (Russo-Neustadt et al., 1999, 2000). Repeated treatments with d-amphetamine (AMPH) have also been reported to increase BDNF levels in multiple brain regions (Meredith et al., 2002). Brain injury induced by experimental stroke or cortical ablation reduces brain levels and/or turnover rates of NE, dopamine (DA) and/or 5-HT for days or weeks after injury (Feeney and Sutton, 1987). Experimental TBI in the rat produces early and widespread reductions in brain NE levels and turnover (Dunn-Meynell et al., 1994; Krobert et al., 1994; Levin et al., 1995). Hence, although exercise in non-injured rats is known to increase NE release (Dunn et al., 1996; Dishman et al., 2000; Garcia et al., 2003), a TBI-induced reduction of NE could conceivably prevent exercise-dependent increases in NE and BDNF during the acute period after injury. Another approach for the treatment of TBI has been to administer treatments that increase central levels of monoamines. Single or multiple treatments with AMPH have been frequently reported to accelerate functional recovery in various brain injury models (Feeney and Sutton, 1988; Sutton and Feeney, 1994), including cortical contusion (Sutton et al., 1987) or FPI (Prasad et al., 1994; Hovda, 1996). While AMPH increases levels of all the mono-amines (Fleckenstein et al., 2007), the beneficial effects of post-injury AMPH treatment appear to be mediated by NE release since intraventricular NE, but not DA (Boyeson and Feeney, 1990) or 5-HT (Boyeson et al., 1994), mimics the AMPH induced recovery of function in ablation models. In addition, drugs causing central NE release induce recovery similar to AMPH (Sutton and Feeney, 1992; Goldstein, 1993), and drugs that interfere with NE release or synaptic transmission can retard recovery or reinstate deficits in recovered animals (Feeney and Westerberg, 1990; Goldstein and Davis, 1990; Dunn-Meynell et al., 1997). In addition to AMPH, multiple treatments with methylphenidate (Kline et al., 2000), a D2 receptor agonist (Kline et al., 2002) or a 5- HT(1A) receptor agonist (Kline et al., 2004) are reported to improve outcome after experimental TBI. More recently, chronic intermittent vagus nerve stimulation, which increases NE release (Roosevelt et al., 2006) and BDNF mRNA levels (Follesa et al., 2007) in cortex and hippocampus, has been shown to improve outcome after experimental TBI (Smith et al., 2005, 2006; Clough et al., 2007). Given these preceding reports on the effects of TBI and the apparent links between AMPH, NE, exercise and BDNF, the current study was designed to determine if AMPH administration during the acute period after TBI would enable or facilitate spontaneous exercise-induced increases of hippocampal BDNF and synapsin I. Because oxidative stress plays an important role in neuronal injury after TBI (Shohami et al., 1997; Sullivan et al., 1998; Hall et al., 2004), we also determined if these treatments would alter oxidative stress after TBI. We employed the controlled cortical impact (CCI) model of TBI for this study, as this injury model is known to produce molecular changes and cellular damage in the hippocampus (Dash et al., 1995; Colicos et al., 1996; Oyesiku et al., 1999; McCullers et al., 2002; Saatman et al., 2006), induce hippocampal-dependent learning deficits (Hamm et al., 1992; Lindner et al., 1998; Dixon et al., 1999), and to reduce brain NE levels and turnover (Dunn-Meynell et al., 1994; Levin et al., 1995). While increased expression or production of hippocampal BDNF and synapsin I has been reported to occur after exercise in both intact rats and in rats with FPI (Timmusk et al., 1993; Neeper et al., 1996; Molteni et al., 2002; Griesbach et al., 2004b, 2007), exercise-induced effects of these markers of plasticity have not yet been evaluated in the CCI model. Based on prior studies we hypothesized that: 1) acute exercise after CCI would have detrimental or neutral effects on outcome measures, 2) low-dose AMPH treatment would improve these outcome measures, and 3) AMPH treatment combined with RW exposure would enable acute exercise to up-regulate BDNF and synapsin I and reduce oxidative stress.

The ability of exercise to benefit neuronal and cognitive plasticity is well recognized. This study reveals that the effects of exercise on brain neuronal and cognitive plasticity are in part modulated by a central source of insulin-like growth factor-I. Exercise selectively increased insulin-like growth factor-I expression without affecting insulin-like growth factor-II expression in the rat hippocampus. To determine the role that insulin-like growth factor-I holds in mediating exercise-induced neuronal and cognitive enhancement, a specific antibody against the insulin-like growth factor-I receptor was used to block the action of insulin-like growth factor-I in the hippocampus during a 5-day voluntary exercise period. A two-trial-per-day Morris water maze was performed for five consecutive days, succeeded by a probe trial 2 days later. Blocking hippocampal insulin-like growth factor-I receptors did not significantly attenuate the ability of exercise to enhance learning acquisition, but abolished the effect of exercise on augmenting recall. Blocking the insulin-like growth factor-I receptor significantly reversed the exercise-induced increase in the levels of brain-derived neurotrophic factor mRNA and protein and pro-brain-derived neurotrophic factor protein, suggesting that the effects of insulin-like growth factor-I may be partially accomplished by modulating the precursor to the mature brain-derived neurotrophic factor. A molecular analysis revealed that exercise significantly elevated proteins downstream to brain-derived neurotrophic factor activation important for synaptic function, i.e. synapsin I, and signal transduction cascades associated with memory processes, i.e. phosphorylated calcium/calmodulin protein kinase II and phosphorylated mitogen-activated protein kinase II. Blocking the insulin-like growth factor-I receptor abolished these exercise-induced increases. Our results illustrate a possible mechanism by which insulin-like growth factor-I interfaces with the brain-derived neurotrophic factor system to mediate exercise-induced synaptic and cognitive plasticity.

Synaptic plasticity and behaviors are likely dependent on the capacity of neurons to meet the energy demands imposed by neuronal activity. We used physical activity, a paradigm intrinsically associated with energy consumption/expenditure and cognitive enhancement, to study how energy metabolism interacts with the substrates for neuroplasticity. We found that in an area critical for learning and memory, the hippocampus, exercise modified aspects of energy metabolism by decreasing oxidative stress and increasing the levels of cytochrome c oxidase-II, a specific component of mitochondrial machinery. We infused 1,25-dihydroxyvitamin D3, a modulator of energy metabolism, directly into the hippocampus during 3 days of voluntary wheel running and measured its effects on brain-derived neurotrophic factor-mediated synaptic plasticity. Brain-derived neurotrophic factor is a central player for the effects of exercise on synaptic and cognitive plasticity. We found that 25-dihydroxyvitamin D3 decreased exercise-induced brain-derived neurotrophic factor but had no significant effect on neurotrophin-3 levels, thereby suggesting a level of specificity for brain-derived neurotrophic factor in the hippocampus. 25-Dihydroxyvitamin D3 injection also abolished the effects of exercise on the consummate end-products of brain-derived neurotrophic factor action, i.e. cyclic AMP response element-binding protein and synapsin I, and modulated phosphorylated calmodulin protein kinase II, a signal transduction cascade downstream to brain-derived neurotrophic factor action that is important for learning and memory. We also found that exercise significantly increased the expression of the mitochondrial uncoupling protein 2, an energy-balancing factor concerned with ATP production and free radical management. Our results reveal a fundamental mechanism by which key elements of energy metabolism may modulate the substrates of hippocampal synaptic plasticity.

A diet high in total fat (HF) reduces hippocampal levels of brain-derived neurotrophic factor (BDNF), a crucial modulator of synaptic plasticity, and a predictor of learning efficacy. We have evaluated the capacity of voluntary exercise to interact with the effects of diet at the molecular level. Animal groups were exposed to the HF diet for 2 months with and without access to voluntary wheel running. Exercise reversed the decrease in BDNF and its downstream effectors on plasticity such as synapsin I, a molecule with a key role in the modulation of neurotransmitter release by BDNF, and the transcription factor cyclic AMP response element binding protein (CREB), important for learning and memory. Furthermore, we found that exercise influenced the activational state of synapsin as well as of CREB, by increasing the phosphorylation of these molecules. In addition, exercise prevented the deficit in spatial learning induced by the diet, tested in the Morris water maze. Furthermore, levels of reactive oxygen species increased by the effects of the diet were decreased by exercise. Results indicate that exercise interacts with the same molecular systems disrupted by the HF diet, reversing their effects on neural function. Reactive oxygen species, and BDNF in conjunction with its downstream effectors on synaptic and neuronal plasticity, are common molecular targets for the action of the diet and exercise. Results unveil a possible molecular mechanism by which lifestyle factors can interact at a molecular level, and provide information for potential therapeutic applications to decrease the risk imposed by certain lifestyles.

This study was designed to identify molecular mechanisms by which exercise affects synaptic-plasticity in the hippocampus, a brain area whose function, learning and memory, depends on this capability. We have focused on the central role that brain-derived neurotrophic factor (BDNF) may play in mediating the effects of exercise on synaptic-plasticity. In fact, this impact of exercise is exemplified by our finding that BDNF regulates the mRNA levels of two end products important for neural function, i.e. cAMP-response-element binding (CREB) protein and synapsin I. CREB and synapsin I have the ability to modify neuronal function by regulating gene-transcription and affecting synaptic transmission, respectively. Furthermore, we show that BDNF is capable of concurrently increasing the mRNA levels of both itself and its tyrosine kinaseB (TrkB) receptor, suggesting that exercise may employ a feedback loop to augment the effects of BDNF on synaptic-plasticity. The use of a novel microbead injection method in our blocking experiments and Taqman reverse transcription polymerase reaction (RT-PCR) for RNA quantification, have enabled us to evaluate the contribution of different pathways to the exercise-induced increases in the mRNA levels of BDNF, TrkB, CREB, and synapsin I. We found that although BDNF mediates exercise-induced hippocampal plasticity, additional molecules, i.e. the N-methyl-D-aspartate receptor, calcium/calmodulin protein kinase II and the mitogen-activated protein kinase cascade, modulate its effects. Since these molecules have a well-described association to BDNF action, our results illustrate a basic mechanism through which exercise may promote synaptic-plasticity in the adult brain.

We have investigated a potential mechanism by which a diet, similar in composition to the typical diet of most industrialized western societies rich in saturated fat and refined sugar (HFS), can influence brain structure and function via regulation of neurotrophins. We show that animals that learn a spatial memory task faster have more brain-derived neurotrophic factor (BDNF) mRNA and protein in the hippocampus. Two months on the HFS diet were sufficient to reduce hippocampal level of BDNF and spatial learning performance. Consequent to the action of BDNF on synaptic function, downstream effectors for the action of BDNF on synaptic plasticity were reduced proportionally to BDNF levels, in the hippocampus of rats maintained on the HFS diet between 2 and 24 months. In particular, animals maintained on the HFS diet showed a decrease in levels of: (i) synapsin I mRNA and protein (total and phosphorylated), important for neurotransmitter release; (ii) cyclic AMP-response element-binding protein (CREB) mRNA and protein (total and phosphorylated); CREB is required for various forms of memory and is under regulatory control of BDNF; (iii) growth-associated protein 43 mRNA, important for neurite outgrowth, neurotransmitter release, and learning and memory. Diet-related changes were specific for the hippocampus consequent to its role in memory formation, and did not involve neurotrophin-3, another member of the neurotrophin family. Our results indicate that a popularly consumed diet can influence crucial aspects of neuronal and behavioral plasticity associated with the function of BDNF.

Previous studies in vitro indicate that basic fibroblast growth factor participates in the survival, proliferation and differentiation of immature neural cells, predicting that it may have the same types of roles in vivo. In order to evaluate a possible role of basic fibroblast growth factor in neural development, we have examined its localization in the rodent brain at critical stages of development. We characterized basic fibroblast growth factor immunoreactivity at embryonic days 13 and 18, and postnatal days 1, 4, 6, 10, 20 and 90. Our results showed that basic fibroblast growth factor was transiently expressed by different cellular phenotypes throughout development. At embryonic day 13, basic fibroblast growth factor immunoreactivity was sparsely distributed in various cell phenotypes. At embryonic day 18, the primitive cerebral cortex showed basic fibroblast growth factor immunoreactivity within its emerging laminar structure, including the cortical plate and subplate regions. At postnatal day 1, basic fibroblast growth factor immunoreactivity was mostly concentrated in the hippocampal subfields cornu Ammon 1, cornu Ammon 2 and cornu Ammon 3, and neurons of the medical septum and the vertical limb of the diagonal band nuclei. At postnatal days 4-6, astrocyte-like cells showed basic fibroblast growth factor immunoreactivity for the first time during development. At this stage, basic fibroblast growth factor in the hippocampus was mostly shown within subfields cornu Ammon 2 and cornu Ammon 1. In the medical septum, just a few neuronal profiles were weakly stained, and basic fibroblast growth factor positive astrocytes appeared to accumulate around these basic fibroblast growth factor-stained neurons. At postnatal day 20, the adult pattern of basic fibroblast growth factor immunoreactivity was fully established. Astrocytes throughout the brain expressed basic fibroblast growth factor, and neuronal basic fibroblast growth factor was restricted to particular populations such as cingulate cortex and hippocampus. The cornu Ammon 2 subfield was the main neuronal location for basic fibroblast growth factor in the mature hippocampus. Our results showed that the cellular location of basic fibroblast growth factor changes during development, suggesting that basic fibroblast growth factor has multiple and evolving roles during histogenesis and differentiation of the CNS.

In order to assess the role of neuronal activity in the regulation of the fibroblast growth factor system, we examined changes in levels of basic fibroblast growth factor and the expression of its receptor-1 following seizures. Epileptiform activity was induced by kainate injection and the rats displaying seizures were killed 3,6, 12 and 24 h after injection. To identify basic fibroblast growth factor and fibroblast growth factor receptor-1 immunoreactivity, we used a monoclonal antibody that binds to the biological active form of basic fibroblast growth factor and a monoclonal antibody that recognizes fibroblast growth factor receptor-1. In normal brain tissue, fibroblast growth factor staining was widely distributed throughout the brain and appeared to be localized within the nucleus of astrocytes. Starting 6 h after seizures, there was a progressive increase in basic fibroblast growth factor immunoreactivity. The seizure-induced effect on basic fibroblast growth factor immunoreactivity was expressed in astrocytes as an enlargement of the nucleus and a spreading of the staining to the processes. This phenomenon was particularly strong in the cerebral cortex and hippocampus. The fibroblast growth factor receptor-1 immunoreactivity was virtually absent in control brain tissue. By 3 h post-seizure induction, there was an increase in fibroblast growth factor receptor-1 immunoreactivity in the molecular layer of the dentate gyrus. After 6 h, fibroblast growth factor receptor-1-positive cells appeared in the stratum oriens along the CA1, CA2 and CA3 hippocampal subfields. This effect gradually expanded to other brain regions and by 24 h fibroblast growth factor receptor-1 immunoreactivity was distributed throughout the hippocampus and cerebral cortex. Fluorescent double labelling indicated that the fibroblast growth factor receptor-1 immunoreactivity was expressed in astrocytes. At 24 h, some fibroblast growth factor receptor-1 immunoreactivity was also observed in neuron-like cells located throughout the cerebral cortex and hippocampus. Since our results indicate that seizure activity modulates the expression of basic fibroblast growth factor and fibroblast growth factor receptor-1 levels, it is also possible that physiological stimulation might have similar effects. In addition, our results suggest that the fibroblast growth factor system may have a role in plasticity events triggered by physiological activity.