Daidzein has neuroprotective effects through ligand-binding-independent PPARγ activation.

Phytoestrogens are a group of plant-derived compounds that include mainly isoflavones like daidzein. Phytoestrogens prevent neuronal damage and improve outcome in experimental stroke; however, the mechanisms of this neuroprotective action have not been fully elucidated. In this context, it has been postulated that phytoestrogens might activate the peroxisome proliferator-activated receptor-γ (PPARγ), which exerts neuroprotective effects in several settings. The aim of this study was to determine whether the phytoestrogen daidzein elicits beneficial actions in neuronal cells by mechanisms involving activation of PPARγ. Our results show that daidzein (0.05-5 µM) decreases cell death induced by exposure to oxygen-glucose deprivation (OGD) from rat cortical neurons and that improves synaptic function, in terms of increased synaptic vesicle recycling at nerve terminals, being both effects inhibited by the PPARγ antagonist T0070907 (1 µM). In addition, this phytoestrogen activated PPARγ in neuronal cultures, as shown by an increase in PPARγ transcriptional activity. Interestingly, these effects were not due to binding to the receptor ligand site, as shown by a TR-FRET PPARγ competitive binding assay. Conversely, daidzein increased PPARγ nuclear protein levels and decreased cytosolic ones, suggesting nuclear translocation. We have used the receptor antagonist (RE) fulvestrant to study the neuroprotective participation of daidzein via estrogen receptor and at least in our model, we have discarded this pathway. These results demonstrate that the phytoestrogen daidzein has cytoprotective properties in neurons, which are due to an increase in PPARγ activity not mediated by direct binding to the receptor ligand-binding domain but likely due to post-translational modifications affecting its subcellular location and not depending to the RE and it is not additive with the agonist rosiglitazone.

"Full moon" endoscopic sign in intraventricular neurocysticercosis.

INTRODUCTION: Despite improvements in sanitation, diagnosis and treatment, neurocysticercosis is still a public health problem in many countries. In symptomatic patients, there is a broad spectrum of clinical manifestations. When cysticerci are lodged in the ventricles or the subarachnoid space, the flow of cerebrospinal fluid can be obstructed and lead to hydrocephalus and intracranial hypertension. The endoscopic view may be useful as a diagnostic tool. PATIENTS: This report clearly shows a common endoscopic pattern in 4 selected patients with ventricular cysticercosis (2 third ventricle/2 lateral ventricle). The endoscopic view of the cysts in the ventricles resembles a "full moon". This analogy helped to identify the features of cysticerci with intact walls and the vesicular stage, malleable due to its cystic content and having an irregular surface, as evidence of the microscopic structure of the cyst wall in a cysticercus. CONCLUSIONS: This finding is not seen in other intraventricular cysts or tumors that can actually be considered as an additional diagnostic criterion among the definitive findings to establish the diagnosis of cysticercosis, since it involves direct endoscopic visualization of a cysticercus under histopathological demonstration. Additionally, the endoscopic approach can be used as primary treatment for these cases, following the minimally invasive approach principle.

The metabotropic glutamate receptor 1 is not involved in the facilitation of glutamate release in cerebrocortical nerve terminals.

In this study we have addressed the identification of the metabotropic glutamate receptor (mGluR) involved in the facilitation of glutamate release in nerve terminals from the cerebral cortex. mGluR1 and 5 are coupled to phosphoinositide hydrolysis and the activation of these receptors with the specific agonist 3,5-dihydroxyphenylglycine (DHPG) enhances the release of glutamate. We have examined whether mGluR1 is responsible for this modulatory effect by preparing nerve terminals from mGluR 1 deficient mice. The Ca2+-dependent glutamate release evoked by a submaximal depolarization is enhanced by the agonist DHPG in nerve terminals from both wild and mutant mice. This result is consistent with the finding that the mGluR agonist also induces a similar increase in the levels of diacylglycerol (DAG) in the nerve terminals from wild and mutant mice. Moreover, the activity-dependent switch from facilitation to inhibition of release, observed when a second stimulation of the receptor is applied shortly after (5 min) the first pulse, was also observed in the mutant mice. These results indicate therefore, that the facilitation of glutamate release is unlikely to be due to the activation of mGluR1 but related to another phosphoinositide coupled mGluR.

Modulation of glutamate release by a nitric oxide/cyclic GMP-dependent pathway.

The mechanism by which changes in cyclic GMP (cGMP) regulate glutamate release was investigated in rat cerebrocortical nerve terminals. The elevation of cGMP levels by inhibition of cGMP-phosphodiesterase with 2-o-propoxy-phenyl-8-azapurin-6-one (zaprinast) reduced the Ca(2+)-dependent glutamate release evoked by depolarization with 30 mM KCl or 1 mM 4-aminopyridine. The nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine also enhanced cGMP and reduced glutamate release. In addition, the membrane-permeable analogs 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and N,2'-o-dibutyrylguanosine (dbcGMP) at 10 microM also mimic glutamate release inhibition. The reduction in glutamate release was observed with no modifications in the ATP/ADP ratio, and was reversed in the presence of the protein kinases inhibitor [N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide, HCl] (H-8). Interestingly, higher concentrations of dbcGMP (1 mM) abolished the inhibition observed with low concentrations although no facilitation was observed. This finding seems to indicate the existence of a dual role for cGMP in the control of glutamate exocytosis.

Presynaptic modulation of glutamate release targets different calcium channels in rat cerebrocortical nerve terminals.

We have studied which type/s of Ca2+-channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCl induced a Ca2+-dependent release of 3.64 +/- 0.25 nmol/mg of protein. The addition of either 2 microM omega-conotoxin-GVIA or 200 nM omega-agatoxin-IVA reduced the KCl-evoked release by 47.7 +/- 3.5% and 70.4 +/- 8.9% respectively, and by 85.7 +/- 4.1% when both toxins were co-applied. The activation of adenosine A1 receptors with N6-cyclohexyladenosine or the activation of metabotropic glutamate receptors with L(+)-2-amino-4-phosphonobutyrate inhibited the KCl-evoked release by 41.0 +/- 5.9 and 54.3 +/- 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 microM omega-conotoxin-GVIA but they were significantly enhanced when omega-agatoxin-IVA was added together with the adenosine A1 receptor agonist or the metabotropic glutamate receptor agonist, suggesting that omega-conotoxin-GVIA-sensitive and not omega-agatoxin-IVA-sensitive Ca2+-channels are involved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C-linked metabotropic receptors, was expressed by both omega-conotoxin-GVIA-sensitive and omega-agatoxin-sensitive Ca2+-channels. It is concluded that different Ca2+-channels support the modulation of glutamate release by presynaptic receptors.

Two components of glutamate exocytosis differentially affected by presynaptic modulation.

The total Ca(2+)-dependent release of glutamate induced by depolarization of cerebrocortical nerve terminals with KCl was analyzed into a fast and a slow component. The fast component exhibited a decay time of < 1 s and accounted for 0.95 +/- 0.10 nmol of glutamate, whereas the slow component, which exhibited a decay time of 52 +/- 7 s, accounted for the release of 2.48 +/- 0.19 nmol of glutamate. These two components were differentially affected by the Ca2+ chelator BAPTA, the divalent cation Sr2+, or the botulinum neurotoxin A. The adenosine A1 receptor agonist N6-cyclohexyladenosine strongly reduced the fast component without altering the slow component. In contrast, the inhibitory effect of arachidonic acid and the facilitatory action of the metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid were observed as a decrease and an increase, respectively, in the two components. It is concluded, first, that the fast and slow components correspond to the release of docked and mobilized vesicles, respectively, and second, that presynaptic modulation more significantly alters the fast component of release.

cAMP-dependent facilitation of glutamate release by beta-adrenergic receptors in cerebrocortical nerve terminals.

We have investigated the presence of a cAMP-protein kinase A-dependent pathway in cerebrocortical nerve terminals and its role in the modulation of glutamate release. The activation of adenylyl cyclase with forskolin enhances intrasynaptosomal cAMP and induces Ca2+-dependent glutamate release. The membrane permeant analogue dibutyryl cAMP mimics this facilitatory effect, whereas the inactive compound 1,9-dideoxyforskolin is without effect. This cAMP-induced facilitation is consistent with the induction of spontaneous action potentials that are abolished by the Na+ channel blocker tetrodotoxin and by reducing nerve terminal excitability with arachidonic acid. We have also demonstrated that a beta-adrenergic receptor is linked to this pathway because isoproterenol increases cAMP levels and glutamate release, and both actions are antagonized by the receptor antagonist propanolol and the protein kinase A inhibitors H89 and 8-chloroadenosine 3',5'-monophosphorothioate ((Rp)-isomer). The finding that the increase in cytoplasmic free Ca2+ concentration induced by synaptic activity reduces the concentration of agonist required to maximally activate adenylyl cyclase suggests that this enzyme may act as a coincidence detector, integrating glutamatergic neurotransmission and noradrenaline release.

Presynaptic receptors and the control of glutamate exocytosis.

When a typical glutamate-containing neurone fires, an action potential is propagated down the branching axon through more than a thousand varicosities. At each of these release sites the probability that a synaptic vesicle will be exocytosed into the synaptic cleft is individually controlled by means of presynaptic receptors: autoreceptors responding by positive or negative feedback to previously released transmitter, or heteroreceptors under the influence of other neurotransmitters or modulators. The simplest system in which to investigate presynaptic modulation is the isolated nerve terminal or synaptosome; studies with this preparation have revealed a complex interplay of signal-transduction pathways.

Decrease in [Ca2+]c but not in cAMP Mediates L-AP4 inhibition of glutamate release: PKC-mediated suppression of this inhibitory pathway.

We investigated the mechanism of the inhibition of glutamate release by (L)-2-amino-4-phosphonobutyrate ((L)-AP4) in cerebrocortical nerve terminals from young rats (3 weeks of age). The Ca(2+)-dependent release of glutamate was reduced by (L)-AP4 in a concentration-dependent manner. This inhibitory effect was prevented by pertussis toxin, insensitive to staurosporine and associated with a reduction both in the depolarization-evoked increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](c)) and in forskolin-stimulated cAMP formation. However, the reduction in [Ca(2+)](c) but not in cAMP seemed to be responsible for the decrease in release, since inhibition by (L)-AP4 can also be observed in the absence of detectable changes in cAMP The inhibitory modulation by (L)-AP4 was suppressed by the activation of protein kinase C with phorbol esters. The nerve terminals from young rats also exhibited a facilitatory pathway of glutamate release which was mediated by protein kinase C. Interestingly, stimulation of this pathway with the glutamate agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in the presence of arachidonic acid also abolished the inhibitory action of (L)-AP4. The dominance of the facilitatory pathway in its interaction with the (L)-AP4-mediated inhibitory control may provide some clues to understand the presynaptic changes during synaptic plasticity.

Co-existence and interaction between facilitatory and inhibitory metabotropic glutamate receptors and the inhibitory adenosine A1 receptor in cerebrocortical nerve terminals.

We have investigated the interaction between facilitatory and inhibitory metabotropic glutamate receptors (mGluRs) and the inhibitory adenosine A1 receptor in cerebrocortical nerve terminals from young (3 weeks postnatal) rats. The adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) (1 microM) and the mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) (100 microM) inhibited Ca(2+)-dependent release of glutamate evoked by depolarization of synaptosomes with 30 mM KCl to 33 +/- 6 and 30 +/- 4% of control values, respectively. The CHA and L-AP4 inhibition of release was consistent with the reduction of a component of Ca2+ entry in nerve terminals which was also sensitive to omega-Aga-IVA. When the inhibitory agonists were co-applied at optimal concentrations, no additivity of the inhibitory effects on either glutamate release or [Ca2+]c was observed. The nerve terminals from young rats also exhibit the facilitatory pathway for glutamate release that is observed during 4-aminopyridine-evoked depolarization after stimulation of mGluRs with the agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) in the presence of arachidonic acid (AA). The addition of ACPD or AA alone did not alter the ability of CHA and L-AP4 to reduce the release, however the co-application of AA and ACPD abolished the inhibitory effect induced by CHA and L-AP4 whether alone or in combination. These results indicate the co-existence of the three modulatory pathways of glutamate release and the dominant role of the ACPD/AA activated facilitatory pathway in its interaction with the inhibitory pathways activated by L-AP4 and CHA.

Rat brain synaptosomes prepared by phase partition.

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.

Occurrence of a large Ca2+-independent release of glutamate during anoxia in isolated nerve terminals (synaptosomes).

Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+-dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+-independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+-independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.

The energy requirement for protein synthesis in rat brain mitochondria purified by phase partition.

Brain mitochondria purified by phase partition showed a higher rate of 14C-leucine incorporation into proteins with an endogenous source of ATP than with an exogenous ATP-generating system. Under the former conditions the presence of atractyloside increased the 14C-leucine incorporation into proteins. The effects of different valinomycin concentrations plus attractyloside on intramitochondrial ATP levels and 14C-leucine incorporation into proteins have been studied. The results indicate that the protein synthesis in brain mitochondria is dependent on the intramitochondrial ATP concentration.

Characterization of the exocytotic release of glutamate from guinea-pig cerebral cortical synaptosomes.

A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca2+-dependent release can be induced not only by K+, but also by the Na+ channel activator veratridine and the Ca2+ ionophore ionomycin. K+-induced release can be inhibited by the Ca2+ channel inhibitor verapamil. Sr2+ and Ba2+ substitute for Ca2+ in promoting K+-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca2+-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast. Ca2+-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.

Neurons from rat forebrain and cerebellum. Isolation and biochemical characterization.

Isolation of neurons from rat forebrain and cerebellum has been performed by a method including trypsin incubation and tissue disaggregation by filtration through successive nylon meshes with a different pore size. Phase contrast microscopy shows highly purified cell preparations. Lactate dehydrogenase, acetylcholinesterase and (Na+-K+) ATPase activities indicate that neurons possess a high cytosolic content and show a good preservation of the plasmatic membrane.