Effect of Hypoproteic and High-Fat Diets on Hippocampal Blood-Brain Barrier Permeability and Oxidative Stress

Worldwide, millions of people are exposed to dietary imbalance that impacts in health and quality of life. In developing countries, like in Brazil, in poor settings, dietary habits, traditionally hypoproteic, are changing rapidly to western-type high-fat foods. These rapidly changing dietary habits are imposing new challenges to human health and there are many questions in the field that remain to be answered. Accordingly, we currently do not know if chronic consumption of hypoproteic (regional basic diet, RBD) or high-fat diets (HFD) may impact the brain physiology, contributing to blood-brain barrier (BBB) dysfunction and neuroinflammatory events. To address this issue, mice were challenged by breastfeeding from dams receiving standard, RBD or HFD from suckling day 10 until weaning. Immediately after weaning, mice continued under the same diets until post-natal day 52. Herein, we show that both RBD and HFD cause not only a peripheral but also a consistent central neuroinflammatory response, characterized by an increased production of Reactive Oxygen Species (ROS) and pro-inflammatory cytokines. Additionally, BBB hyperpermeability, accounted by an increase in hippocampal albumin content, a decrease in claudin-5 protein levels and collagen IV immunostaining, was also observed together with an upregulation of vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we also identified a significant astrogliosis, manifested by upregulation of GFAP and S100β levels and an intensification of arbor complexity of these glial cells. In sum, our data show that dietary imbalance, related with hypoproteic or high-fat content, impairs BBB properties potentially favoring the transmigration of peripheral immune cells and induces both a peripheral and central neuroinflammatory status. Noteworthy, neuroinflammatory events in the hippocampus may cause neuronal malfunction leading to cognitive deficits and long-term persistence of this phenomenon may contribute to age-related neurodegenerative diseases

Understanding the physiology of glutamate receptors by use of a protocol for neuronal staining

Teaching students about the physiology of neurotransmitter receptors usually requires practical lessons with the use of sophisticated equipment and complex analysis of data. Here, we report our experience in teaching medical students with a simple, practical protocol that transforms the physiology of glutamate receptors into neuronal staining, observable under bright-field microscopy. Essentially, the students were challenged to selectively stain a subpopulation of cultured neurons expressing Ca(2+)-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (a subgroup of ionotropic glutamate receptors). Neurons expressing this type of receptors were loaded with Co(2+) (in substitution for Ca(2+)) after nondesensitizing activation of AMPA receptors. After precipitation, the Co(2+) was revealed after treatment with silver. At the end of the procedure, the neurons expressing Ca(2+)-permeable AMPA receptors were visually identified under bright-field microscopy. The procedure allowed the visualization of the complete dendritic network of the stained neurons and allowed the students to learn very efficiently about the physiology of glutamate receptor

Adult Hippocampal Neurogenesis in Different Taxonomic Groups: Possible Functional Similarities and Striking Controversies

Adult neurogenesis occurs in many species, from fish to mammals, with an apparent reduction in the number of both neurogenic zones and new neurons inserted into established circuits with increasing brain complexity. Although the absolute number of new neurons is high in some species, the ratio of these cells to those already existing in the circuit is low. Continuous replacement/addition plays a role in spatial navigation (migration) and other cognitive processes in birds and rodents, but none of the literature relates adult neurogenesis to spatial navigation and memory in primates and humans. Some models developed by computational neuroscience attribute a high weight to hippocampal adult neurogenesis in learning and memory processes, with greater relevance to pattern separation. In contrast to theories involving neurogenesis in cognitive processes, absence/rarity of neurogenesis in the hippocampus of primates and adult humans was recently suggested and is under intense debate. Although the learning process is supported by plasticity, the retention of memories requires a certain degree of consolidated circuitry structures, otherwise the consolidation process would be hampered. Here, we compare and discuss hippocampal adult neurogenesis in different species and the inherent paradoxical aspects

Protein kinase C activity blocks neuropeptide Y-mediated inhibition of glutamate release and contributes to excitability of the hippocampus in status epilepticus

The unbalanced excitatory/inhibitory neurotransmitter function in the neuronal network afflicted by seizures is the main biochemical and biophysical hallmark of epilepsy. The aim of this work was to identify changes in the signaling mechanisms associated with neuropeptide Y (NPY)-mediated inhibition of glutamate release that may contribute to hyperexcitability. Using isolated rat hippocampal nerve terminals, we showed that the KCl-evoked glutamate release is inhibited by NPY Y2 receptor activation and is potentiated by the stimulation of protein kinase C (PKC). Moreover, we observed that immediately after status epilepticus (6 h postinjection with kainate, 10 mg/kg), the functional inhibition of glutamate release by NPY Y2 receptors was transiently blocked concomitantly with PKC hyperactivation. The pharmacological blockade of seizure-activated PKC revealed again the Y2 receptor-mediated inhibition of glutamate release. The functional activity of PKC immediately after status epilepticus was assessed by evaluating phosphorylation of the AMPA receptor subunit GluR1 (Ser-831), a substrate for PKC. Moreover, NPY-stimulated [35S]GTPgammaS autoradiographic binding studies indicated that the common target for Y2 receptor and PKC on the inhibition/potentiation of glutamate release was located downstream of the Y2 receptor, or its interacting G-protein, and involves voltage-gated calcium channel

Neuroprotective effect of H. perforatum extracts on β-amyloid-induced neurotoxicity

12 pages, 6 figures, 1 table.In the present study we assessed the neuroprotective role of aHypericum perforatum ethanolic extract and obtained fractions in amyloid-β peptide (Aβ)(25–35)-induced cell death in rat cultured hippocampal neurons. Lipid peroxidation was used as a marker of oxidative stress by following the formation of TBARS in rat cortical synaptosomes, after incubation with ascorbate/Fe2+, alone or in the presence of EC97 effective concentrations ofH. perforatum fractions. Induced lipid peroxidation was significantly inhibited by fractions containing flavonol glycosides, flavonol and biflavone aglycones, and by a fraction containing several phenols, mainly chlorogenic acid-type phenolics (21%,77%and 98%, respectively). Lipid peroxidation evaluated after incubation with 25 μM Aβ(25–35), was significantly inhibited byH. perforatum extract. Cell viability was assessed by use of the Syto-13/PI assay. The total ethanolic extract (TE) and fractions containing flavonol glycosides, flavonol and biflavone aglycones, reduced Aβ(25–35)-induced cell death (65%,58%and 59%,respectively). These results were further supported by morphological analysis of cells stained with cresyl violet. Peptide β-amyloid(25–35) induced a decrease in cell volume, chromatin condensation and nuclear fragmentation, alterations not evident in the presence of the TE and fractions containing hypericins (hypericin concentration = 11.02 μM), or fractions containing flavonoids (quercetin concentration = 21.13 μM). Dendritic lesion,an evidence of neurodegeneration, was observed by neuronal staining with cobalt following insult with Aβ(25–35), but prevented after exposure to the peptide plus the fractions referred above. The results of the present paper suggest thatH. perforatum extracts may be endowed with neuroprotective compounds able to prevent Aβ(25–35)-induced toxicity.This work was supported by the Fundação de Ciência e Tecnologia (POCTI/AGR /40283/2001 project) and GRICES (Proc. 423 ICCTI/CSIC). B. Silva is supported by Fundação para a Ciência e a Tecnologia with a Ph.D. grant (SFRH/BD/13488/2003).Peer reviewe

Up-regulation of neuropeptide Y levels and modulation of glutamate release through neuropeptide Y receptors in the hippocampus of kainate-induced epileptic rats

Kainate-induced epilepsy has been shown to be associated with increased levels of neuropeptide Y (NPY) in the rat hippocampus. However, there is no information on how increased levels of this peptide might modulate excitation in kainate-induced epilepsy. In this work, we investigated the modulation of glutamate release by NPY receptors in hippocampal synaptosomes isolated from epileptic rats. In the acute phase of epilepsy, a transient decrease in the efficiency of NPY and selective NPY receptor agonists in inhibiting glutamate release was observed. Moreover, in the chronic epileptic hippocampus, a decrease in the efficiency of NPY and the Y2 receptor agonist, NPY13-36, was also found. Simultaneously, we observed that the epileptic hippocampus expresses higher levels of NPY, which may account for an increased basal inhibition of glutamate release. Consistently, the blockade of Y2 receptors increased KCl-evoked glutamate release, and there was an increase in Y2 receptor mRNA levels 30 days after kainic acid injection, suggesting a basal effect of NPY through Y2 receptors. Taken together, these results indicate that an increased function of the NPY modulatory system in the epileptic hippocampus may contribute to basal inhibition of glutamate release and control hyperexcitability

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors

Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures

The inflammatory cytokines interleukin-1\u2424 and tumor necrosis factor-\u2423 (TNF-\u2423) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic hippocampal slice cultures to toxic concen- trations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-\u2423 (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-\u2423 to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-\u2423 receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-\u2423 involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine- producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic condition