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We have previously demonstrated that exposure of adult rat to a type of enriched environment, known as ‘naturalistic habitat’ (NH), induces extensive functional plasticity in the whiskers’ representations within the primary somatosensory cortex. Here we have investigated the molecular basis for such functional plasticity involved in this model. Based on the role of BDNF on synaptic plasticity and neuronal growth, the focus of this study is on BDNF and its downstream effectors CREB, synapsin I, and GAP-43. In particular, we determined the effects of natural whiskers use during 2, 7 or 28 days exposure to a NH on barrel cortex and hippocampus, as compared to standard cage controls. Naturalistic whiskers use resulted in increased levels of mRNAs and proteins for BDNF and its downstream effectors. Level changes for these markers were already detected after 2 days in the naturalistic habitat and grew larger over longer exposures (7 and 28 days). The cerebral cortex was found to be sensitive to the naturalistic habitat exposure at all time points, and more sensitive than the hippocampus to the trimming of the whiskers. Trimming of the whiskers decreased the level of most of the markers under study suggesting that whiskers exert a tonic influence on plasticity markers that can be further enhanced by naturalistic use. These results implicate BDNF and its downstream effectors in the plasticity induced by the naturalistic habitat. The critical action of experience on molecular substrates of plasticity seems to provide molecular basis for the design of experienced-based rehabilitative strategies to enhance brain function.

We report that rats learning a spatial memory task in the Morris water maze show elevated expression of the signal transduction receptor for BDNF and the synaptic associated protein synapsin I in the hippocampus. Nuclease protection assays showed maximal levels of TrkB and synapsin I mRNAs in the hippocampus by the time that asymptotic learning performance had been reached (Day 6). Increases in synapsin I mRNA were matched by changes in synapsin I protein as revealed by western blot analysis. Synapsin I is a downstream effector for the BDNF tyrosine kinase cascade pathway which has important roles in synaptic remodeling and function. Therefore, parallel changes in TrkB and synapsin I mRNAs suggest a role of the BDNF system in synaptic function or adaptation. Levels of TrkB mRNA in the hippocampus were attenuated after learning acquisition (Day 20), but synapsin I mRNA was still elevated, suggesting that the BDNF system may participate in events secondary to learning, such as strengthening of neural circuits. TrkB and synapsin I mRNAs showed an increasing trend in the cerebellum of learning rats and no changes were observed in the caudal cerebral cortex. The selectivity of the changes in trkB and synapsin I, affecting the hippocampus, is in agreement with the role of this structure in processing of spatial information. Behavioral regulation of neurotrophins may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to promote neural healing after CNS injury or disease.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) support the viability and function of many types of neurons, and are likely mediators of activity-dependent changes in the CNS. We examined BDNF and NGF mRNA levels in several brain areas of adult male rats following 0, 2, 4, or 7 nights with ad libitum access to running wheels. BDNF mRNA was significantly increased in several brain areas, most notably in the hippocampus and caudal 1/3 of cerebral cortex following 2, 4, and 7 nights with exercise. Significant elevations in BDNF mRNA were localized in Ammon's horn areas 1 (CA1) and 4 (CA4) of the hippocampus, and layers II-III of the caudal neocortex and retrosplenial cortex. NGF mRNA was also significantly elevated in the hippocampus and caudal 1/3 of the cortex, affecting primarily the dentate gyrus granular layer (DG) and CA4 of the hippocampus and layers II-III in caudal neocortex.

Dietary omega-3 fatty acid (i.e. docosohexaenoic acid (DHA)) and exercise are gaining recognition for supporting brain function under normal and challenging conditions. Here we evaluate the possibility that the interaction of DHA and exercise can involve specific elements of the synaptic plasma membrane. We found that voluntary exercise potentiated the effects of a 12-day DHA dietary supplementation regimen on increasing the levels of syntaxin 3 (STX-3) and the growth-associated protein (GAP-43) in the adult rat hippocampus region. STX-3 is a synaptic membrane-bound protein involved in the effects of DHA on membrane expansion. The DHA diet and exercise also elevated levels of the NMDA receptor subunit NR2B, which is important for synaptic function underlying learning and memory. The actions of exercise and DHA dietary supplementation reflected on enhanced learning performance in the Morris water maze as learning ability was associated with higher levels of STX-3 and NR2B. The overall findings reveal a mechanism by which exercise can interact with the function of DHA dietary enrichment to elevate the capacity of the adult brain for axonal growth, synaptic plasticity, and cognitive function.

We have previously shown that voluntary exercise upregulates brain derived neurotrophic factor (BDNF) within the hippocampus and is associated with an enhancement of cognitive recovery after a lateral fluid percussion injury (FPI). In order to determine if BDNF is critical to this effect we used an immunoadhesin chimera (TrkB-IgG) that inactivates free BDNF. This BDNF inhibitor was administered to adult male rats two weeks after they had received a mild fluid percussion injury (FPI) or sham surgery. These animals were then housed with or without access to a running wheel (RW) from post-injury-day (PID) 14 to 20. On PID 21, rats were tested for spatial learning in a Morris Water Maze. Results showed that exercise counteracted the cognitive deficits associated with the injury. However this exercise-induced cognitive improvement was attenuated in the FPI-RW rats that were treated with TrkB-IgG. Molecules important for synaptic plasticity and learning were measured in a separate group of rats that were sacrificed immediately after exercise (PID 21). Western blot analyses showed that exercise increased the mature form of BDNF, synapsin I and cyclic-AMP response-element-binding protein (CREB) in the vehicle treated Sham-RW group. However, only the mature form of BDNF and CREB were increased in the vehicle treated FPI-RW group. Blocking BDNF (pre administration of TrkB-IgG) greatly reduced the molecular effects of exercise in that exercise-induced increases of BDNF, synapsin I and CREB were not observed. These studies provide evidence that BDNF has a major role in exercise's cognitive effects in traumatically injured brain.

Following traumatic brain injury (TBI), the brain undergoes a period of metabolic and neurochemical alterations that may compromise the reactivity of neuroplasticity-related molecular systems to physiological stimulation. In order to address the molecular mechanisms underlying plasticity following TBI and the effects of physical stimulation in the acute phase of TBI, levels of intracellular signaling molecules were assessed following voluntary exercise. Lateral fluid percussion injury (FPI) and sham-operated (Sham) rats were housed with or without access to a running wheel (RW) from postsurgery day 0 to 6. Parietal and occipital cortical tissues were analyzed for brain-derived neurotrophic factor (BDNF) using an enzyme-linked immunoabsorbant assay (ELISA). In addition, synapsin I, phospho-synapsin I, cyclic-AMP response-element-binding protein (CREB), phospho-CREB, calcium-calmodulin-dependent protein kinase II (CAMKII), mitogen-activated protein (MAP) kinase I and II (MAPKI and MAPKII), and protein kinase C (PKC) were analyzed by western blot. Results from this study indicated that FPI alone lead to significant increases in synapsin I, CAMKII, and phosphorylated (P) MAPKI (p44) and MAPKII (p42). Exercise in the sham operates led to significant cortical increases of CREB and synapsin I. However, in the FPI rats, the response to exercise was opposite to that seen in the shams in that exercise resulted in significant decreases of CREB, synapsin I, PKC, CAMKII, MAPKI, and MAPKII. Indeed, all the observed proteins in the acutely exercised FPI rats tended to be lower compared to the FPI sedentary (Sed) rats. These results indicate that intracellular signaling proteins are increased during the first week following FPI and that premature voluntary exercise may compromise plasticity.

We have evaluated changes in the expression of neurotrophin-3 (NT-3) and its tyrosine kinase C (TrkC) receptor in the neuromuscular system as a result of voluntary physical activity. We assessed changes in the mRNAs and proteins for NT-3 and TrkC in the lumbar spinal cord and associated soleus muscle following 3 and 7 days of voluntary wheel running. We used quantitative Taqman RT-PCR to measure mRNA and ELISA to assess protein levels. NT-3 mRNA and protein levels increased in the spinal cord to reach statistical significance after 7 days of exercise compared to sedentary control rats. Immunohistochemical analyses localized the elevated NT-3 to the substantia gelatinosa (SG) and nucleus of the dorsal horn. TrkC mRNA levels were significantly elevated in the spinal cord after 3 and 7 days of running. In the soleus muscle, NT-3 mRNA levels and its receptor TrkC were elevated after 3 days, while NT-3 protein levels remained unaffected. The results demonstrate that voluntary exercise has a differential effect on NT-3 as well as its receptor TrkC in the neural and muscular components of the neuromuscular system, and emphasize the role of voluntary activity on the spinal cord and muscle.

Clinical and experimental evidence indicate that physical activity has a positive impact on brain function; however, the molecular bases for how exercise affects the structure and function of the brain are largely unknown. We have investigated the influences of variable periods of voluntary wheel-running on the expression of basic fibroblast growth factor and its mRNA in various brain regions. Nuclease protection assays revealed that the hippocampus was the only region examined exhibiting changes in FGF-2 mRNA as a result of exercise. FGF-2 mRNA increased to reach a peak by the 4th night of wheel-running. FGF-2 immunoreactivity, normally located in the perinuclear area of astrocytes, following exercise became stronger and appeared to spread to the cytoplasm and processes of astrocytes. Quantification of the FGF-2-immunoreactive astrocytes showed an increase in density between 2 and 4 nights of running in discrete regions of the hippocampus. These results demonstrate that exercise regulates FGF-2 expression and suggest that growth factors are likely mediators of the positive effects of exercise on the brain.

We studied remodeling of the remaining corticorubral projections in adult cats sustaining a left cerebral hemispherectomy in adulthood or neonatally using cortical injections of [3H]leucine-proline. Injection sites and terminal fields were reconstructed from autoradiography-processed tissue. In all cats, the label filled similar extents of areas 4γ and 3a of the right frontal cortex. We used sections at 8 coronal planes throughout the red nucleus (RN) for computer-assisted analysis of visually estimated density and topography of distribution of terminal label, and for calculation of RN cross-sectional area. Additionally, at 3 coronal planes we further quantified terminal label using computerized procedures (number of particles for surface area). In all lesioned cats we found terminal label in the RN contralateral to the injection site with a topographic distribution similar to that of the RN ipsilateral to the injection in normal or lesioned cats and in absence of any significant shrinkage of the nucleus. The difference between the 2 age-at-lesion groups was that in the cats with neonatal ablation the density of contralateral terminal label was about double that seen in adult-lesioned subjects. However, the amount of contralateral labeling in adult-lesioned cats was substantial and represented a significant increase over the minimal labeling seen in normal cats. There were no differences between groups in labeling or size of the RN ipsilateral to the injection site. For reasons discussed, we interpret the label on the side of the hemispherectomy as representative of reinnervation of the cortically deafferented RN by crossing collaterals of fibers arising in the remaining motor cortex and not as lesion-sustained persistent prenatal connections.

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.

This is a quantitative study of changes in distribution and density of terminals of the corticospinal tract in the cervical spinal cord and dorsal column nuclei (DCN) in cats with left cerebral hemispherectomy performed neonatally or in adulthood. Kittens received hemispherectomy at a mean of 12.1 postnatal days and were compared, as adults, to adult-lesioned cats of similar survival time. All animals, including controls, received injections of [3H]leucine-proline and were sacrificed 5 days later. Injection sites and terminal fields were reconstructed from autoradiography-processed tissue. The label filled comparable extents of areas 4 gamma and 3a of the right cerebral cortex. Coronal sections from upper and lower cervical cord levels, and from the brainstem (cuneate and gracile nuclei) were studied. Computer-image processing procedures were used to count labeled particles from multiple sites of the dorsal horn and DCN, bilaterally. In the spinal cord of intact and adult-hemispherectomized cats, most terminals were found in lamina VI, and adjacent laminae V and VII contralateral to the injection side. The major finding was that neonatal-lesioned cats showed a significant increase in axon terminals in areas ipsilateral to the injection. The topography of distribution of the novel terminals was similar to that in the contralateral side and the originating fibers appeared to have crossed the midline from that side. A similar reorganization occurred in the gracile nucleus where, in intact and adult-lesioned cats, the cortical terminals also predominated in the side contralateral to the injection. In contrast, neonatal-lesioned animals showed a significant increase in terminal density ipsilateral to the cortical injection. These findings are discussed as an alternative mechanism for postlesion remodeling of the corticospinal tract in animals with the pyramidal crossing completed at the time of birth.

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.