Forebrain Homogenates Research Articles

Pharmacological Characterization of [18F]-FNM and Evaluation of NMDA Receptors Activation in a Rat Brain Injury Model

PurposeNMDA receptors (NMDARs) dysfunction plays a central role in the physiopathology of psychiatric and neurodegenerative disorders whose mechanisms are still poorly understood. The development of a PET (positron emission tomography) tracer able to selectively bind to the NMDARs intra-channel PCP site may make it possible to visualize NMDARs in an open and active state. We describe the in vitro pharmacological characterization of [18F]-fluoroethylnormemantine ([18F]-FNM) and evaluate its ability to localize activated NMDA receptors in a rat preclinical model of excitotoxicity.ProceduresThe affinity of the non-radioactive analog for the intra-channel PCP site was determined in a radioligand competition assay using [3H]TCP ([3H]N-(1-[thienyl]cyclohexyl)piperidine) on rat brain homogenates. Selectivity was also investigated by the displacement of specific radioligands targeting various cerebral receptors. In vivo brain lesions were performed using stereotaxic quinolinic acid (QA) injections in the left motor area (M1) of seven Sprague Dawley rats. Each rat was imaged with a microPET/CT camera, 40 min after receiving a dose of 30 MBq + / − 20 of [18F]-FNM, 24 and 72 h after injury. Nine non-injured rats were also imaged using the same protocol.ResultsFNM displayed IC50 value of 13.0 ± 8.9 µM in rat forebrain homogenates but also showed significant bindings on opioid receptors. In the frontal and left somatosensory areas, [18F]FNM PET detected a mean of 37% and 41% increase in [18F]FNM uptake (p < 0,0001) 24 and 72 h after QA stereotaxic injection, respectively, compared to the control group.ConclusionsIn spite of FNM’s poor affinity for NMDAR PCP site, this study supports the ability of this tracer to track massive activation of NMDARs in neurological diseases.

Extrasynaptic δ-GABAA receptors are high-affinity muscimol receptors.

Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal non‐selective GABA‐site agonist. Deletion of the GABAA receptor (GABAAR) δ subunit in mice (δKO) leads to a drastic reduction in high‐affinity muscimol binding in brain sections and to a lower behavioral sensitivity to muscimol than their wild type counterparts. Here, we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a > 50% loss of high‐affinity 5 nM [3H]muscimol‐binding sites despite the relatively low abundance of δ‐containing GABAARs (δ‐GABAAR) in the brain. By subtracting residual high‐affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δ‐GABAARs in WT mice exhibit high‐affinity [3H]muscimol‐binding sites (KD ~1.6 nM on α4βδ receptors in the forebrain and ~1 nM on α6βδ receptors in the cerebellum at 22°C). Co‐expression of the δ subunit with α6 and β2 or β3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3H]muscimol component. In addition, we compared muscimol currents in recombinant α4β3δ and α4β3 receptors and show that δ subunit co‐expression leads to highly muscimol‐sensitive currents with an estimated EC 50 of around 1–2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABAAR muscimol sensitivity. We conclude that biochemical and behavioral low‐dose muscimol selectivity for δ‐subunit‐containing receptors is a result of low nanomolar‐binding affinity on δ‐GABAARs.

Pharmacologic Characterization of Valbenazine (NBI-98854) and Its Metabolites.

The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2, is approved for the treatment of tardive dyskinesia. Valbenazine is converted to two significant circulating metabolites in vivo, namely, (+)-α-dihydrotetrabenazine (R,R,R-HTBZ) and a mono-oxy metabolite, NBI-136110. Radioligand-binding studies were conducted to assess and compare valbenazine, tetrabenazine, and their respective metabolites in their abilities to selectively and potently inhibit [3H]-HTBZ binding to VMAT2 in rat striatal, rat forebrain, and human platelet homogenates. A broad panel screen was conducted to evaluate possible off-target interactions of valbenazine, R,R,R-HTBZ, and NBI-136110 at >80 receptor, transporter, and ion channel sites. Radioligand binding showed R,R,R-HTBZ to be a potent VMAT2 inhibitor in homogenates of rat striatum (Ki = 1.0-2.8 nM), rat forebrain (Ki = 4.2 nM), and human platelets (Ki = 2.6-3.3 nM). Valbenazine (Ki = 110-190 nM) and NBI-136110 (Ki = 160-220 nM) also exhibited inhibitory effects on VMAT2, but with lower potency than R,R,R-HTBZ. Neither valbenazine, R,R,R-HTBZ, nor NBI-136110 had significant off-target interactions at serotonin (5-HT1A, 5-HT2A, 5-HT2B) or dopamine (D1 or D2) receptor sites. In vivo studies measuring ptosis and prolactin secretion in the rat confirmed the specific and dose-dependent interactions of tetrabenazine and R,R,R-HTBZ with VMAT2. Evaluations of potency and selectivity of tetrabenazine and its pharmacologically active metabolites were also performed. Overall, the pharmacologic characteristics of valbenazine appear consistent with the favorable efficacy and tolerability findings of recent clinical studies [KINECT 2 (NCT01733121), KINECT 3 (NCT02274558)].

Cholinergic Abnormalities, Endosomal Alterations and Up-Regulation of Nerve Growth Factor Signaling in Niemann-Pick Type C Disease

BackgroundNeurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF.ResultsNPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A.ConclusionsOur results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.

Dispersible amyloid β-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice

Soluble amyloid β-protein (Aβ) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aβ aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aβ aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aβ-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aβ oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aβ and Aβ plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aβ oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aβ oligomers, protofibrils, and fibrils represent an important pool of Aβ aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aβ aggregates, thereby, presumably determines its toxicity.

On the antioxidant properties of kynurenic acid: Free radical scavenging activity and inhibition of oxidative stress

Kynurenic acid (KYNA) is an endogenous metabolite of the kynurenine pathway for tryptophan degradation and an antagonist of both N-methyl- d-aspartate (NMDA) and alpha-7 nicotinic acetylcholine (α7nACh) receptors. KYNA has also been shown to scavenge hydroxyl radicals ( OH) under controlled conditions of free radical production. In this work we evaluated the ability of KYNA to scavenge superoxide anion (O 2 −) and peroxynitrite (ONOO −). The scavenging ability of KYNA (expressed as IC 50 values) was as follows: OH = O 2 − > ONOO −. In parallel, the antiperoxidative and scavenging capacities of KYNA (0–150 μM) were tested in cerebellum and forebrain homogenates exposed to 5 μM FeSO 4 and 2.5 mM 3-nitropropionic acid (3-NPA). Both FeSO 4 and 3-NPA increased lipid peroxidation (LP) and ROS formation in a significant manner in these preparations, whereas KYNA significantly reduced these markers. Reactive oxygen species (ROS) formation were determined in the presence of FeSO 4 and/or KYNA (0–100 μM), both at intra and extracellular levels. An increase in ROS formation was induced by FeSO 4 in forebrain and cerebellum in a time-dependent manner, and KYNA reduced this effect in a concentration-dependent manner. To further know whether the effect of KYNA on oxidative stress is independent of NMDA and nicotinic receptors, we also tested KYNA (0–100 μM) in a biological preparation free of these receptors – defolliculated Xenopus laevis oocytes – incubated with FeSO 4 for 1 h. A 3-fold increase in LP and a 2-fold increase in ROS formation were seen after exposure to FeSO 4, whereas KYNA attenuated these effects in a concentration-dependent manner. In addition, the in vivo formation of OH evoked by an acute infusion of FeSO 4 (100 μM) in the rat striatum was estimated by microdialysis and challenged by a topic infusion of KYNA (1 μM). FeSO 4 increased the striatal OH production, while KYNA mitigated this effect. Altogether, these data strongly suggest that KYNA, in addition to be a well-known antagonist acting on nicotinic and NMDA receptors, can be considered as a potential endogenous antioxidant.

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

Fragile X syndrome is the most common heritable form of mental retardation. It is caused by silencing of the Fmr1 gene and the absence of the encoded protein. The purpose of this study was to examine global protein expression levels of GABA(A) and GABA(B) receptors, and GABAergic enzymes and trafficking proteins in fragile X knockout mice during brain maturation. Quantitative western blotting of homogenates of forebrain revealed that the levels of GABA(A) beta1 and beta3, GABA(B)-R1, NKCC1, KCC2, gephyrin and ubiquilin were not significantly different from wild-type mice at any of the postnatal time points examined. In contrast, the GABA(A) receptor alpha1, beta2, and delta subunits, and the GABA enzymes GABA transaminase and succinic semialdehyde dehydrogenase were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse brain. The GABA(A) receptor alpha1 and beta2 subunits displayed a divergent pattern of developmental expression whereby alpha1 was reduced in the immature brain but regained a level of expression similar to wild-type mice by adulthood, while the expression of beta2 was similar to wild-types at postnatal day 5 but reduced at day 12 and in the adult brain. The GABA(A) receptor delta subunit and the GABA catabolic enzymes GABA transaminase and succinic semialdehyde dehydrogenase were simultaneously but transiently decreased only at postnatal day 12. Our results demonstrate that GABA(A) receptor subunits and GABA enzymes display complex patterns of changes during brain development suggesting that dynamic interactions may occur between GABA transmitter levels and GABA receptors in fragile X syndrome.

Distribution of a novel binding site for angiotensins II and III in mouse tissues

A novel binding site for angiotensins II and III that is unmasked by parachloromercuribenzoate has been reported in rat, mouse and human brains. Initial studies of this binding site indicate that it is not expressed in the adrenal, liver or kidney of the rat and mouse. To determine if this binding site occurs in other mouse tissues, 8 tissues were assayed for expression of this binding site by radioligand binding assay and compared with the expression of this binding site in the forebrain. Particulate fractions of homogenates of testis, epididymis, seminal vesicles, heart, spleen, pancreas, lung, skeletal muscle, and forebrain were incubated with 125I-sarcosine 1, isoleucine 8 angiotensin II in the presence or absence of 0.3 mM parachloromercuribenzoate plus 10 µM losartan and 10 µM PD123319 (to saturate AT 1 and AT 2 receptors). Specific (3 µM angiotensin II displaceable) high affinity binding occurred in the testis > forebrain > epididymis > spleen > pancreas > lung when parachloromercuribenzoate was present. Binding could not be reliably observed in heart, skeletal muscle and seminal vesicles. High affinity binding of 125I-sarcosine 1, isoleucine 8 angiotensin II was observed in the absence of parachloromercuribenzoate in the pancreas on occasion. This suggests that this novel angiotensin binding site may have a functional role in these tissues.

Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo

Our aim was to determine whether a Vaccinium myrtillus (bilberry) anthocyanoside (VMA) and/or its main anthocyanidin constituents (cyanidin, delphinidin, and malvidin) can protect retinal ganglion cells (RGCs) against retinal damage in vitro and in vivo. In RGC cultures (RGC-5, a rat ganglion cell-line transformed using E1A virus) in vitro, cell damage and radical activation were induced by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Cell viability was measured using a water-soluble tetrazolium salt assay. Intracellular radical activation within RGC-5 cells was evaluated using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA). Lipid peroxidation was assessed using the supernatant fraction of mouse forebrain homogenates. In mice in vivo, we evaluated the effects of VMA on N-methyl-D-aspartic acid (NMDA)-induced retinal damage using hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stainings. VMA and all three anthocyanidins (i) significantly inhibited SIN-1-induced neurotoxicity and radical activation in RGC-5, (ii) concentration-dependently inhibited lipid peroxidation in mouse forebrain homogenates. Intravitreously injected VMA significantly inhibited the NMDA-induced morphological retinal damage and increase in TUNEL-positive cells in the ganglion cell layer. Thus, VMA and its anthocyanidins have neuroprotective effects (exerted at least in part via an anti-oxidation mechanism) in these in vitro and in vivo models of retinal diseases.

Influence of Memantine on Brain Monoaminergic Neurotransmission Parameters in Mice: Neurochemical and Behavioral Study

Memantine, a non-competitive antagonist of NMDA receptors, has recently been used in Alzheimer's disease. The influences of memantine on behavioral changes, monoamine oxidase (MAO) activity and reuptake of both serotonin (5-HT) and dopamine in mice were examined in the present study. Memantine dose-dependently increased locomotor activity. This effect was inhibited by intraperitoneal (i.p.) administration of haloperidol. Furthermore, administration [intracerebroventricular (i.c.v.)] of memantine did not induce the head-twitch response (HTR). However, the 5-HT-induced HTR was potentiated by the combined administration of memantine. The enhanced HTR was inhibited by i.p. administration of haloperidol or 5-HT(2A) antagonist ketanserin. Memantine at 1 mM inhibited both MAO-A and MAO-B activities in mouse forebrain homogenates to 37% and 64% of controls, respectively. Lineweaver-Burk plots analysis revealed competitive inhibition with both MAO-A and MAO-B. The inhibitions were also reversible. Memantine inhibited the reuptake of both 5-HT and dopamine into mouse forebrain synaptosomes. 5-HT and dopamine reuptakes were inhibited to 2% and 16% of controls, respectively, with 1 mM memantine. These findings suggest that the increased locomotor activity and enhanced 5-HT-induced HTR by memantine may result from the reuptake and turnover inhibitions of 5-HT and dopamine.

Anticholinergic Activity of 107 Medications Commonly Used by Older Adults

The objective of this study was to measure the anticholinergic activity (AA) of medications commonly used by older adults. A radioreceptor assay was used to investigate the AA of 107 medications. Six clinically relevant concentrations were assessed for each medication. Rodent forebrain and striatum homogenate was used with tritiated quinuclidinyl benzilate. Drug-free serum was added to medication and atropine standard-curve samples. For medications that showed detectable AA, average steady-state peak plasma and serum concentrations (C(max)) in older adults were used to estimate relationships between in vitro dose and AA. All results are reported in pmol/mL of atropine equivalents. At typical doses administered to older adults, amitriptyline, atropine, clozapine, dicyclomine, doxepin, L-hyoscyamine, thioridazine, and tolterodine demonstrated AA exceeding 15 pmol/mL. Chlorpromazine, diphenhydramine, nortriptyline, olanzapine, oxybutynin, and paroxetine had AA values of 5 to 15 pmol/mL. Citalopram, escitalopram, fluoxetine, lithium, mirtazapine, quetiapine, ranitidine, and temazepam had values less than 5 pmol/mL. Amoxicillin, celecoxib, cephalexin, diazepam, digoxin, diphenoxylate, donepezil, duloxetine, fentanyl, furosemide, hydrocodone, lansoprazole, levofloxacin, metformin, phenytoin, propoxyphene, and topiramate demonstrated AA only at the highest concentrations tested (patients with above-average C(max) values, who receive higher doses, or are frail may show AA). The remainder of the medications investigated did not demonstrate any AA at the concentrations examined. Psychotropic medications were particularly likely to demonstrate AA. Each of the drug classifications investigated (e.g., antipsychotic, cardiovascular) had at least one medication that demonstrated AA at therapeutic doses. Clinicians can use this information when choosing between equally efficacious medications, as well as in assessing overall anticholinergic burden.

Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain

We have characterized the expression of microRNAs and selected microRNA precursors within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated post-synaptic densities (PSDs), using methods of microRNA microarray, real time qRT-PCR, Northern blotting and immunopurification using anti-PSD95 antibody. The majority of brain microRNAs (especially microRNAs known to be expressed in pyramidal neurons) are detectably expressed in synaptic fractions, and a subset of microRNAs is significantly enriched in synaptic fractions relative to total forebrain homogenate. MicroRNA precursors are also detectable in synaptic fractions at levels that are comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors are predominantly associated with PSDs. For seven microRNAs examined, there was a significant correlation between the relative synaptic enrichment of the precursor and the relative synaptic enrichment of the corresponding mature microRNA. These findings support the proposal that microRNAs are formed, at least in part, via processing of microRNA precursors locally within dendritic spines. Dicer is expressed in PSDs but is enzymatically inactive until conditions that activate calpain cause its liberation; thus, we propose that synaptic stimulation may lead to local processing of microRNA precursors in proximity to the synapse.

Synaptic enrichment of microRNAs in adult mouse forebrain is related to structural features of their precursors

Within mouse forebrain, a subset of microRNAs are significantly enriched in synaptoneurosomes (a synaptic fraction containing pinched-off dendritic spines) and a subset are significantly depleted relative to total forebrain homogenate. Here I show that, as a group, the pre-miR hairpin precursors of synaptically enriched microRNAs exhibit significantly different structural features than those that are non-enriched or depleted. Precursors of synaptically enriched microRNAs tend to have a) shorter uninterrupted double-stranded stem segments, and b) more symmetrical bulges containing a single nucleotide on each side. These structural differences may provide a basis for the differential binding of proteins that mediate dendritic transport of pre-miRs, or that prevent pre-miRs from being prematurely processed into mature miRNAs during the transport process.This article was reviewed by I. King Jordan and Jerzy Jurka.

Kalirin-7 Controls Activity-Dependent Structural and Functional Plasticity of Dendritic Spines

Activity-dependent rapid structural and functional modifications of central excitatory synapses contribute to synapse maturation, experience-dependent plasticity, and learning and memory and are associated with neurodevelopmental and psychiatric disorders. However, the signal transduction mechanisms that link glutamate receptor activation to intracellular effectors that accomplish structural and functional plasticity are not well understood. Here we report that NMDA receptor activation in pyramidal neurons causes CaMKII-dependent phosphorylation of the guanine-nucleotide exchange factor (GEF) kalirin-7 at residue threonine 95, regulating its GEF activity, leading to activation of small GTPase Rac1 and rapid enlargement of existing spines. Kalirin-7 also interacts with AMPA receptors and controls their synaptic expression. By demonstrating that kalirin expression and spine localization are required for activity-dependent spine enlargement and enhancement of AMPAR-mediated synaptic transmission, our study identifies a signaling pathway that controls structural and functional spine plasticity.

BRAG1, a Sec7 domain-containing protein, is a component of the postsynaptic density of excitatory synapses

The postsynaptic density (PSD) at excitatory synapses is a dynamic complex of glutamatergic receptors and associated proteins that governs synaptic structure and coordinates signal transduction. In this study, we report that BRAG1, a putative guanine nucleotide exchange factor for the Arf family of GTP-binding proteins, is a major component of the PSD. BRAG1 was identified in a 190 kDa band in the PSD fraction with the use of mass spectrometry coupled to searching of a protein sequence database. BRAG1 expression is abundant in the adult rat forebrain, and it is strongly enriched in the PSD fraction compared to forebrain homogenate and synaptosomes. Immunocytochemical localization of BRAG1 in dissociated hippocampal neurons shows that it forms discrete clusters that colocalize with the postsynaptic marker PSD-95 at sites along dendrites. BRAG1 contains a Sec7 domain, a domain that catalyzes exchange of GDP for GTP on the Arf family of small GTP-binding proteins. In their GTP-bound active state, Arfs regulate trafficking of vesicles and cytoskeletal structure. We demonstrate that the Sec7 domain of BRAG1 promotes binding of GTP to Arf in vitro. These data suggest that BRAG1 may modulate the functions of Arfs at synaptic sites.

Water extract of propolis and its main constituents, caffeoylquinic acid derivatives, exert neuroprotective effects via antioxidant actions

We investigated whether water extract of Brazilian green propolis (WEP) and its main constituents [caffeoylquinic acid derivatives (3,4-di- O-caffeoylquinic acid, 3,5-di- O-caffeoylquinic acid, chlorogenic acid) and cinnamic acid derivatives ( p-coumaric acid, artepillin C, drupanin, baccharin)] exert neuroprotective effects against the retinal damage induced by oxidative stress. Additionally, their neuroprotective effects were compared with their antioxidant effects. WEP, 3,4-di- O-caffeoylquinic acid, 3,5-di- O-caffeoylquinic acid, chlorogenic acid, and p-coumaric acid (but not artepillin C, baccharin, or drupanin) concentration-dependently inhibited oxidative stress-induced neurotoxicity [achieved using l-buthionine-( S, R)-sulfoximine (BSO) to deplete glutathione in combination with glutamate to inhibit cystine uptake] in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus). At their effective concentrations against oxidative stress-induced retinal damage, WEP, 3,4-di-caffeoylquinic acid, 3,5-di-caffeoylquinic acid, and chlorogenic acid (but not cinnamic acid derivatives) inhibited lipid peroxidation (LPO) in mouse forebrain homogenates. Thus, the neuroprotective effects of WEP and caffeoylquinic acid derivatives paralleled those against LPO. These findings indicate that WEP and caffeoylquinic acid derivatives have neuroprotective effects against retinal damage in vitro, and that these effects may be partly mediated via antioxidant effects.

Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion - induced oxidative stress products formation in rat forebrain.

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe(2+)+H(2)O(2)). In animals subjected to four-vessel global ischemia for 15 min and 2-24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.

Allosteric modulation of 5-HT 3 serotonin receptors

[ 3H]Granisetron binding to 5-HT 3 type serotonin receptors was examined in homogenates of rat forebrain and NG 108-15 cells. We have applied an allosteric model to 5-HT 3 receptor binding for the first time. Slope factors of displacement improved the modelling. Serotonin displaced [ 3H]granisetron binding with micromolar potency in forebrain and with nanomolar potency in NG 108-15 cells. Racemic and (+)verapamil, ifenprodil and GYKI-46903 were used as representative allosteric inhibitors of 5-HT 3 receptors. They displaced [ 3H]granisetron binding with great negative cooperativity ( α > 10) and exerted great negative cooperativity with serotonin binding ( β > 10). Great negative cooperativity of these agents with serotonin and [ 3H]granisetron binding cannot be distinguished from dual competitive displacement. Trichloroethanol (data from literature) had no cooperativity with [ 3H]granisetron binding ( α ~ 1) and exhibit positive cooperativity with serotonin ( β < 1) in displacement. The allosteric model can lead to a more quantitative method in vitro to develop allosteric agents for 5-HT 3 receptors.

Minocycline inhibits oxidative stress and decreases in vitro and in vivo ischemic neuronal damage

The neuroprotective effects of minocycline–which is broadly protective in neurologic-disease models featuring cell death and is being evaluated in clinical trials–were investigated both in vitro and in vivo. For the in vivo study, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice. Minocycline at 90 mg/kg intraperitoneally administered 60 min before or 30 min after (but not 4 h after) the occlusion reduced infarction, brain swelling, and neurologic deficits at 24 h after the occlusion. For the in vitro studies, we used cortical-neuron cultures from rat fetuses in which neurotoxicity was induced by 24-h exposure to 500 μM glutamate. Furthermore, the effects of minocycline on oxidative stress [such as lipid peroxidation in mouse forebrain homogenates and free radical-scavenging activity against diphenyl- p-picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. Minocycline significantly inhibited glutamate-induced cell death at 2 μM and lipid peroxidation and free radical scavenging at 0.2 and 2 μM, respectively. These findings indicate that minocycline has neuroprotective effects in vivo against permanent focal cerebral ischemia and in vitro against glutamate-induced cell death and that an inhibition of oxidative stress by minocycline may be partly responsible for these effects.

Neuroprotection by Brazilian Green Propolis against In vitro and In vivo Ischemic Neuronal Damage

We examined whether Brazilian green propolis, a widely used folk medicine, has a neuroprotective function in vitro and/or in vivo. In vitro, propolis significantly inhibited neurotoxicity induced in neuronally differentiated PC12 cell cultures by either 24 h hydrogen peroxide (H2O2) exposure or 48 h serum deprivation. Regarding the possible underlying mechanism, propolis protected against oxidative stress (lipid peroxidation) in mouse forebrain homogenates and scavenged free radicals [induced by diphenyl-p-picrylhydrazyl (DPPH). In mice in vivo, propolis [30 or 100 mg/kg; intraperitoneally administered four times (at 2 days, 1 day and 60 min before, and at 4 h after induction of focal cerebral ischemia by permanent middle cerebral artery occlusion)] reduced brain infarction at 24 h after the occlusion. Thus, a propolis-induced inhibition of oxidative stress may be partly responsible for its neuroprotective function against in vitro cell death and in vivo focal cerebral ischemia.