Lúcia Vinadé

Exercise affects glutamate receptors in postsynaptic densities from cortical mice brain

Physical activity has been proposed as a behavior intervention that promotes mental health and some of the benefits induced by exercise have been related to the glutamatergic system. Indeed, glutamate is the most abundant excitatory neurotransmitter in brain. Thus, we evaluated if voluntary exercise in mice could modulate glutamatergic synapses at level of postsynaptic density (PSD). Through Western blot, we found that exercise during 1 month increased glutamatergic-related protein content in PSD from cortex of mice. Exercise increased the immunocontent of GluR1 (129%), SAP-97 (179%), GRIP-1 (129%), and in less extent, GluR2/3 (118%) and PSD-95 (112%) proteins. The overall content of NMDA subunits R1, R2A and R2B were not altered in mice that had exercised, however, the phosphorylated NMDA subunits, phospho-NMDAR1 (150%), and phospho-NMDAR2B (183%) showed a strong increase. Because exercise increased the content of phosphorylated forms of NMDA receptors, we evaluated the binding of MK-801, a specific ligand that binds to open NMDA channel. Exercise increased the binding of MK-801 in cortical cellular membranes in 51%. Altogether, our results point to a modulation of glutamatergic synapses by exercise with likely implications in the exercise-induced mental health.

Developmental Changes in S100B Content in Brain Tissue, Cerebrospinal Fluid, and Astrocyte Cultures of Rats

Abstract1. We investigated the content of S100B protein by ELISA in three brain regions (hippocampus, cerebral cortex, and cerebellum) and in cerebrospinal fluid of rats during postnatal development as well as the content and secretion of S100B in pre- and postconfluent primary astrocyte cultures.2. An accumulation of S100B occurred in all brain regions with similar ontogenetic pattern between second and fourth postnatal weeks. However, we observed a decrease in the cerebrospinal fluid S100B after the critical period for synaptogenesis in rodents.3. A similar profile of cell accumulation and decrease in basal secretion was also observed during aging of astrocyte cultures.4. These data contribute to the proposal that S100B is an important glial-derived protein during brain development and that changes in extracellular levels of S100B may be related to glial proliferation and synaptogenesis.

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: relevance for behavior alterations.

Essential omega-3 polyunsaturated fatty acids (omega3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary omega3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with omega3 (omega3 group) or a omega3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (alphaCaMKII) were evaluated. Additionally, hippocampal [(3)H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the omega3 group, although the difference disappeared at 21 days of life (except for alphaCaMKII, which content normalized at 60 days old). The same pattern was found for [(3)H]glutamate binding, whereas [(3)H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary omega3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary omega3 deficiency is discussed.

Guanine and adenine nucleotidase activities in rat cerebrospinal fluid

Adenine and guanine nucleotides have been shown to exert multiple roles in central and peripheral nervous systems, and the sequential breakdown of these nucleotides by enzymatic systems is an important step in the modulation of their extracellular effects. The aim of this study was to investigate whether nucleotide hydrolysis also occurs in the cerebrospinal fluid (CSF) of rats. CSF was able to hydrolyze all guanine and adenine nucleotides investigated (2.0 mM): GDPz.Gt;ADP=ATP=GTPz.Gt;AMP=GMP. More detailed studies with the diphosphate nucleotides showed that the hydrolysis of ADP and GDP was linear with incubation time and protein concentration. The apparent K(M) (Henry-Michaelis-Menten constant) and V (maximal velocity) values for ADP and GDP were 164.3+/-54.7 microM and 12.2+/-3.8 nmol P(i)/min per mg protein, and 841.0+/-90.2 microM and 22.8+/-8.0 nmol P(i)/min per mg protein. The sum of ADP, GDP and UDP hydrolysis (2.0 mM) upon individual incubations with CSF was similar to the hydrolysis observed when all three nucleotides were incubated together. This pattern of hydrolysis strongly suggests the involvement of more than one enzyme activity. The higher maximum activity for GDP and UDP compared to ADP is compatible with presence of a soluble NTDPase5.

Dietary omega-3 fatty acids attenuate cellular damage after a hippocampal ischemic insult in adult rats ☆

The role of omega-3 polyunsaturated fatty acids (3PUFAs) on brain function is increasingly demonstrated. Here, the effect of dietary deprivation of essential 3PUFAs on some parameters related to neuroprotection was investigated. Rats were fed with two different diets: omega-3 diet and omega-3-deprived diet. To assess the influence of 3PUFAs on brain responses to ischemic insult, hippocampal slices were subjected to an oxygen and glucose deprivation (OGD) model of in vitro ischemia. The omega-3-deprived group showed higher cell damage and stronger decrease in the [(3)H]glutamate uptake after OGD. Moreover, omega-3 deprivation influenced antiapoptotic cell response after OGD, affecting GSK-3beta and ERK1/2, but not Akt, phosphorylation. Taken together, these results suggest that 3PUFAs are important for cell protection after ischemia and also seem to play an important role in the activation of antiapoptotic signaling pathways.

Evidence for a role for calcium ions in the dephosphorylation of glial fibrillary acidic protein (GFAP) in immature hippocampal slices and in astrocyte cultures from the rat

Evidence was sought for a role for Ca2+ in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in immature hippocampal slices. Although previous work showed that the main phosphatase dephosphorylating GFAP in this preparation is a Ca2+-independent type 1 enzyme, a role for Ca2+ was suggested by the observation that the incorporation of [32P]phosphate into GFAP in immature slices is inhibited by external Ca2+. This inhibition is strikingly different to the situation in mature slices where GFAP phosphorylation is completely dependent on Ca2+. Pure astrocyte cultures were probed by immunoblotting for the presence of the Ca2+-dependent phosphatase calcineurin. An enzyme content, amounting to about 2% of that found in fresh hippocampal tissue, was detected for both the catalytic (alpha) and regulatory (beta) subunits. The direct or indirect association of calcineurin with GFAP was suggested by observations showing that FK506, a specific inhibitor of calcineurin, increased the phosphorylation state of GFAP in immature slices and of GFAP and vimentin in astrocyte cultures.

Dietary omega-3 deficiency reduces BDNF content and activation NMDA receptor and Fyn in dorsal hippocampus: Implications on persistence of long-term memory in rats

Abstract Omega-3 (n-3) fatty acids are important for adequate brain function and cognition. The aim of the present study was to evaluate how n-3 fatty acids influence the persistence of long-term memory (LTM) in an aversive memory task and to explore the putative mechanism involved. Female rats received isocaloric diets that included n-3 (n-3 group) or not (D group). The adult litters were subjected to an inhibitory avoidance task (0.7 mA, 1.0 seconds foot shock) to elicit persistent LTM. Twelve hours after the training session, the fatty acid profile and the brain derived neurotrophic factor (BDNF) content of the dorsal hippocampus were assessed. In addition, we measured the activation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor and the SRC family protein Fyn. Despite pronounced learning in both groups, the persistence of LTM was abolished in the D group 7 days after the training session. We also observed that the D group presented reductions in hippocampal DHA (22:6 n-3) and BDNF content. Twelve hours after the training session, the D group showed decreased NR2B and Fyn phosphorylation in the dorsal hippocampus, with no change in the total content of these proteins. Further, there was a decrease in the interaction of Fyn with NR2B in the D group, as observed by co-immunoprecipitation. Taken together, these data suggest that n-3 fatty acids influence the persistence of LTM by maintaining adequate levels of DHA and BDNF as well as by influencing the activation of NR2B and Fyn during the period of memory formation.

Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus.

The effect of glutamate and lack of external Ca2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12-16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517-523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181-186), glutamate strongly stimulated GFAP phosphorylation in media with Ca2+ and in media lacking Ca2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices.

The dephosphorylation of glial fibrillary acidic protein (GFAP) in the immature rat hippocampus is catalyzed mainly by a type 1 protein phosphatase.

We used protein phosphatase inhibitors to study the phosphatase activity involved in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in a cytoskeletal fraction and in slices prepared from hippocampi of immature rats. Cytoskeletal proteins were labelled with [gamma 32P]ATP in the presence of Ca2+ and calmodulin and then allowed to dephosphorylate through the activity of bound protein phosphatases. Dephosphorylation was inhibited by the protein phosphatase inhibitors okadaic acid and microcystin-LR, but not by EGTA. Maximal inhibition was given by 1 microM okadaic acid and 10 nM microcystin-LR. This difference of two orders of magnitude in the sensitivity of the dephosphorylation to the inhibitors indicates that the bound dephosphorylating activity in the cytoskeletal fraction was due to a type 1 protein phosphatase, rather than protein phosphatase 2A which is equally sensitive to okadaic acid and microcystin-LR. To investigate the dephosphorylation of GFAP in intact tissue we incubated slices with various concentrations of the cell-permeable inhibitor okadaic acid in the presence of [32P] phosphate. Net [32P]-incorporation into GFAP was increased by okadaic acid due to inhibition of dephosphorylation; the minimum effective concentration was 25 nM. Since the IC50 for inhibition of protein phosphatase 2A by okadaic acid is 0.1 nM, this result indicates that in intact tissue GFAP dephosphorylation is primarily due to a type 1 protein phosphatase.

Short-term alterations in hippocampal glutamate transport system caused by one-single neonatal seizure episode: implications on behavioral performance in adulthood.

Impairment in the activity and expression of glutamate transporters has been found in experimental models of epilepsy in adult animals. However, there are few studies investigating alterations on glutamate transporters caused by epilepsy in newborn animals, especially in the early periods after seizures. In this study, alterations in the hippocampal glutamate transporters activity and immunocontent were investigated in neonatal rats (7 days old) submitted to kainate-induced seizures model. Glutamate uptake, glutamate transporters (GLT-1, GLAST, EAAC1) and glutamine synthetase (GS) were assessed in hippocampal slices obtained 12 h, 24 h, 48 h, 72 h and 60 days after seizures. Immunoreactivity for hippocampal GFAP, NeuN and DAPI were assessed 24 h after seizure. Behavioral analysis (elevated-plus maze and inhibitory avoidance task) was also investigated in the adult animals (60 days old). The decrease on glutamate uptake was observed in hippocampal slices obtained 24 h after seizures. The immunocontent of GLT-1 increased at 12 h and decreased at 24 h (+62% and -20%, respectively), while GLAST increased up to 48 h after seizures. No alterations were observed for EAAC1 and GS. It should be mentioned that there were no long-term changes in tested glutamate transporters at 60 days after kainate treatment. GFAP immunoreactivity increased in all hippocampal subfields (CA1, CA3 and dentate gyrus) with no alterations in NeuN and DAPI staining. In the adulthood, kainate-treated rats showed anxiety-related behavior and lower performance in the inhibitory avoidance task. Our findings indicate that acute modifications on hippocampal glutamate transporters triggered by a single convulsive event in early life may play a role in the behavioral alterations observed in adulthood.