Caroline C. Real

Short-term, moderate exercise is capable of inducing structural, BDNF-independent hippocampal plasticity.

Exercise is known to improve cognitive functions and to induce neuroprotection. In this study we used a short-term, moderate intensity treadmill exercise protocol to investigate the effects of exercise on usual markers of hippocampal synaptic and structural plasticity, such as synapsin I (SYN), synaptophysin (SYP), neurofilaments (NF), microtubule-associated protein 2 (MAP2), glutamate receptor subunits GluR1 and GluR2/3, brain-derived neurotrophic factor (BDNF) and glial fibrillary acidic protein (GFAP). Immunohistochemistry, Western blotting and real-time PCR were used. We also evaluated the number of cells positive for the proliferation marker 5-bromo-2-deoxyuridine (BrdU), the neurogenesis marker doublecortin (DCX) and the plasma corticosterone levels. Adult male Wistar rats were adapted to a treadmill and divided into 4 groups: sedentary (SED), 3-day exercise (EX3), 7-day exercise (EX7) and 15-day exercise (EX15). The protein changes detected were increased levels of NF68 and MAP2 at EX3, of SYN at EX7 and of GFAP at EX15, accompanied by a decreased level of GluR1 at EX3. Immunohistochemical findings revealed a similar pattern of changes. The real-time PCR analysis disclosed only an increase of MAP2 mRNA at EX7. We also observed an increased number of BrdU-positive cells and DCX-positive cells in the subgranular zone of the dentate gyrus at all time points and increased corticosterone levels at EX3 and EX7. These results reveal a positive effect of short-term, moderate treadmill exercise on hippocampal plasticity. This effect was in general independent of transcriptional processes and of BDNF upregulation, and occurred even in the presence of increased corticosterone levels.

BDNF RECEPTOR BLOCKADE HINDERS THE BENEFICIAL EFFECTS OF EXERCISE IN A RAT MODEL OF PARKINSON'S DISEASE

Physical exercise is known to produce beneficial effects to the nervous system. In most cases, brain-derived neurotrophic factor (BDNF) is involved in such effects. However, little is known on the role of BDNF in exercise-related effects on Parkinsons disease (PD). The aim of this study was to investigate the effects of intermittent treadmill exercise-induced behavioral and histological/neurochemical changes in a rat model of unilateral PD induced by striatal injection of 6-hydroxydopamine (6-OHDA), and the role of BDNF in the exercise effects. Adult male Wistar rats were divided into two main groups: (1) injection of K252a (a blocker of BDNF receptors), and (2) without BDNF receptor blockade. These groups were then subdivided into four groups: control (CLT), sedentary (SED, non-exercised with induction of PD), exercised 3×/week during four weeks before and four weeks after the induction of PD (EXB+EXA), and exercised 3×/week during four weeks after the induction of PD (EXA). One month after 6-OHDA injections, the animals were subjected to rotational behavioral test induced by apomorphine and the brains were collected for immunohistochemistry and immunoblotting assays, in which we measured BDNF and tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) and the striatum (caudate-putamen, CPu). Our results showed a significant reduction of rotational asymmetry induced by apomorphine in the exercised parkinsonian rats. BDNF decreased in the SNc of the SED group, and exercise was able to revert that effect. Exercised groups exhibited reduced damage to the dopaminergic system, detected as a decreased drop of TH levels in SNc and CPu. On the other hand, BDNF blockade was capable of substantially reducing TH expression postlesion, implying enhanced dopaminergic cell loss. Our data revealed that physical exercise is capable of reducing the damage induced by 6-OHDA, and that BDNF receptors are involved in that effect.

Exercise-induced plasticity of AMPA-type glutamate receptor subunits in the rat brain.

The aim of this study was to analyze the plastic effects of moderate exercise upon the motor cortex (M1 and M2 areas), cerebellum (Cb), and striatum (CPu) of the rat brain. This assessment was made by verifying the expression of AMPA-type glutamate receptor subunits (GluR1 and GluR2/3). We used adult Wistar rats, divided into 5 groups based on duration of exercise training, namely 3 days (EX3), 7 days (EX7), 15 days (EX15), 30 days (EX30), and sedentary (S). The exercised animals were subjected to a treadmill exercise protocol at the speed of the 10 meters/min for 40 min. After exercise, the brains were subjected to immunohistochemistry and immunoblotting to analyze changes of GluR1 and GluR2/3, and plasma corticosterone was measured by ELISA in order to verify potential stress induced by physical training. Overall, the results of immunohistochemistry and immunoblotting were similar and revealed that GluR subunits show distinct responses over the exercise periods and for the different structures analyzed. In general, there was increased expression of GluR subunits after longer exercise periods (such as EX30), although some opposite effects were seen after short periods of exercise (EX3). In a few cases, biphasic patterns with decreases and subsequent increases of GluR expression were seen and may represent the outcome of exercise-dependent, complex regulatory processes. The data show that the protocol used was able to promote plastic GluR changes during exercise, suggesting a specific involvement of these receptors in exercise-induced plasticity processes in the brain areas tested.

Moderate exercise changes synaptic and cytoskeletal proteins in motor regions of the rat brain.

Physical exercise is known to enhance brain function in several aspects. We evaluated the acute effects of a moderate forced exercise protocol on synaptic proteins, namely synapsin I (SYN) and synaptophysin (SYP), and structural proteins (neurofilaments, NFs) in rat brain regions related to motor function and often affected by neurodegenerative disorders. Immunohistochemistry, Western blotting and real-time PCR were used to analyze the expression of those proteins after 3, 7 and 15days of exercise (EX3, EX7 and EX15). In the cerebellum, increase of SYN was observed at EX7 and EX15 and of NF68 at EX3. In the substantia nigra, increases of protein levels were observed for NF68 and NF160 at EX3. In the striatum, there was an increase of SYN at EX3 and EX7, of SYP at EX7 and of NF68 at EX3. In the cortex, decreased levels of NF68 and NF160 were observed at EX3, followed by an increase of NF68 at EX15. In the reticular formation, all NF proteins were increased at EX15. The mRNA data for each time-point and region also revealed significant exercise-related changes of SYN, SYP and NF expression. These results suggest that moderate physical exercise modulates synaptic and structural proteins in motor brain areas, which may play an important role in the exercise-dependent brain plasticity.

Different protocols of physical exercise produce different effects on synaptic and structural proteins in motor areas of the rat brain

The plastic brain responses generated by the training with acrobatic exercise (AE) and with treadmill exercise (TE) may be different. We evaluated the protein expression of synapsin I (SYS), synaptophysin (SYP), microtubule-associated protein 2 (MAP2) and neurofilaments (NF) by immunohistochemistry and Western blotting in the motor cortex, striatum and cerebellum of rats subjected to TE and AE. Young adult male Wistar rats were divided into 3 groups: sedentary (Sed) (n=15), TE (n=20) and AE (n=20). The rats were trained 3 days/week for 4 weeks on a treadmill at 0.6 km/h, 40 min/day (TE), or moved through a circuit of obstacles 5 times/day (AE). The rats from the TE group exhibited a significant increase of SYS and SYP in the motor cortex, of NF68, SYS and SYP in the striatum, and of MAP2, NF and SYS in the cerebellum, whereas NF was decreased in the motor cortex and the molecular layer of the cerebellar cortex. On the other hand, the rats from the AE group showed a significant increase of MAP2 and SYP in the motor cortex, of all four proteins in the striatum, and of SYS in the cerebellum. In conclusion, AE induced changes in the expression of synaptic and structural proteins mainly in the motor cortex and striatum, which may underlie part of the learning of complex motor tasks. TE, on the other hand, promoted more robust changes of structural proteins in all three regions, especially in the cerebellum, which is involved in learned and automatic tasks.

REACTIVE OXYGEN SPECIES AND THE STRUCTURAL REMODELING OF THE VISUAL SYSTEM AFTER OCULAR ENUCLEATION

Redox processes associated with controlled generation of reactive oxygen species (ROS) by NADPH oxidase (Nox) add an essential level of regulation to signaling pathways underlying physiological processes. We evaluated the ROS generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation in adult rats. Dihydroethidium (DHE) oxidation revealed increased ROS generation in SC and DLG between 1 and 30 days postlesion. ROS generation was decreased by the Nox inhibitors diphenyleneiodonium chloride (DPI) and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment. Taken together, the findings of the present study suggest a novel role for Nox-induced ROS signaling in mediating neuronal remodeling in visual areas after ocular enucleation.

Different protocols of treadmill exercise induce distinct neuroplastic effects in rat brain motor areas

A variety of exercise protocols have been used to promote experimental neuroplasticity. However, the plastic brain responses generated by several aspects of training (types, frequency, regimens, duration) remain undetermined. The aim of this study was to compare the plastic changes in the glutamatergic system and synaptic proteins in motor cortex, striatum and cerebellum promoted by two different treadmill exercise regimens. The present study analyzed by immunohistochemistry and Western blotting the expression of the subunits of AMPA receptors (GluA1 and GluA2/3) and synaptic proteins (synapsin I and synaptophysin) in adult male Wistar rat brains. The animals were divided into animals subjected to two different frequencies of aerobic exercise groups and sedentary animals. The exercise groups were: intermittent treadmill exercise (ITE) - animals that exercised 3 times a week (every other day) during four weeks, and continuous treadmill exercise (CTE) - animals that exercised every day during four weeks. Our results reveal that different protocols of treadmill exercise were able to promote distinct synaptic reorganization processes among the exercised groups. In general, the intermittent exercise regimen induced a higher expression of presynaptic proteins, whereas the continuous exercise regimen increased postsynaptic GluA1 and GluA2/3 receptors.

Temporal Changes of CB1 Cannabinoid Receptor in the Basal Ganglia as a Possible Structure-Specific Plasticity Process in 6-OHDA Lesioned Rats

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration, neuroprotection and neuronal plasticity. The CB1 cannabinoid receptors are abundantly expressed in the basal ganglia, the circuitry that is mostly affected in Parkinson’s Disease (PD). Some studies show variation of CB1 expression in basal ganglia in different animal models of PD, however the results are quite controversial, due to the differences in the procedures employed to induce the parkinsonism and the periods analyzed after the lesion. The present study evaluated the CB1 expression in four basal ganglia structures, namely striatum, external globus pallidus (EGP), internal globus pallidus (IGP) and substantia nigra pars reticulata (SNpr) of rats 1, 5, 10, 20, and 60 days after unilateral intrastriatal 6-hydroxydopamine injections, that causes retrograde dopaminergic degeneration. We also investigated tyrosine hydroxylase (TH), parvalbumin, calbindin and glutamic acid decarboxylase (GAD) expression to verify the status of dopaminergic and GABAergic systems. We observed a structure-specific modulation of CB1 expression at different periods after lesions. In general, there were no changes in the striatum, decreased CB1 in IGP and SNpr and increased CB1 in EGP, but this increase was not sustained over time. No changes in GAD and parvalbumin expression were observed in basal ganglia, whereas TH levels were decreased and the calbindin increased in striatum in short periods after lesion. We believe that the structure-specific variation of CB1 in basal ganglia in the 6-hydroxydopamine PD model could be related to a compensatory process involving the GABAergic transmission, which is impaired due to the lack of dopamine. Our data, therefore, suggest that the changes of CB1 and calbindin expression may represent a plasticity process in this PD model.

Ontogeny of subunits 2 and 3 of the AMPA-type glutamate receptors in Purkinje cells of the developing chick cerebellum.

Several molecules, involved in cellular communication in the mature nervous system, appear to play important roles during neural development. These roles include neuronal growth, morphological changes of neurites, and neuronal survival. Such plasticity processes seem to be in part the result of activation of different receptor subtypes, which could cause Ca(2+) influx, a major candidate to be an outgrowth promoter. In this context, we performed immunohistochemical and in situ hybridization experiments to examine the following aspects of the development of chick cerebellum Purkinje cells: (i) expression of AMPA-type glutamate receptor GluR2/3 proteins; (ii) the levels of mRNAs coding for the GluR2 and GluR3 flip/flop isoforms; and (iii) expression of calbindin (CB) and parvalbumin (PV). Expression of GluR2/3 proteins, CB, PV, and the mRNAs coding for GluR2 and GluR3 splice variants all revealed a differential expression during development in chick Purkinje cells. GluR2/3 proteins and the GluR3 flop variant start to be expressed at E10, while the expression of CB, PV, the GluR3 flip isoform and the splice variants of GluR2 all started around E12-E14. All proteins showed an increasing expression from embryonic stages into the posthatching period. These results reveal a developmentally regulated expression of GluR2/3 proteins, including their splice variants, and of CB and PV in Purkinje cells. These findings may suggest a relationship between these proteins and specific cerebellar developmental processes.

Differential response of AMPA and NMDA glutamate receptors of Purkinje cells to aging of the chicken cerebellum

Aging can lead to cognitive, affective, learning, memory and motor deficits. Since the cerebellum and glutamatergic neurotransmission are involved in several of those functions, the present work aimed at studying the expression of AMPA and NMDA glutamate receptor subunits in the chick cerebellum during aging. Young (30 days old) and aged (ca. 4 years old) chickens (Gallus gallus) were used in order to evaluate the expression of GluR1, GluR2/3 and NR1 subunits. The cerebella of young and aged chickens were subjected to immunohistochemical and immunoblotting techniques. Numbers of GluR1, GluR2/3 and NR1-positive cells and optical density of the immunoblotting data were analyzed and submitted to statistical analysis using ANOVA and the Bonferroni post hoc test. Mean density of Purkinje cells stained for Giemsa, GluR1, GluR2/3 and NR1 in the cerebellum all showed a statistically significant decrease in aged animals when compared to the young animals (Giemsa, P<0.01; GluRs and NR1, P<0.03). However, the ratio of GluR1 and GluR2/3-positive Purkinje cells in relation the total number of Purkinje cells found in each time point decreased with aging (ca. 10%), whereas the ratio of NR1-positive cells increased (ca. 9%). The immunoblotting data showed a significant decrease of GluR1 (ca. 66%) and GluR2/3 (ca. 55%) protein expression with aging, but did not reveal changes for NR1. Our data suggest that aging can lead to differential changes in the pattern of expression of glutamate receptor subunits, which can underlie at least part of the cognitive and motor disorders found in aged animals.