A monoamine oxidase B gene variant and short-term antidepressant treatment response

Genetic differences among patients suffering from Major Depression are likely to contribute to interindividual differences in medication treatment response. Thus, the identification of gene variants affecting drug response is needed in order to be able to predict response to psychopharmacological drugs. This study analyzed a possible association of the common A644G single nucleotide polymorphism (SNP) within intron 13 of the monoamine oxidase B (MAOB) gene with antidepressant treatment response. The study population consisted of n = 102 patients with major depression (criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition; DSM-IV) participating in a randomized double-blind controlled clinical trial, conducted at 50 centers in Germany, comparing the efficacy of mirtazapine and paroxetine during 6 weeks of treatment. Overall, female patients homozygous for the A-allele had a significantly faster and more pronounced antidepressant treatment response than AG/GG-carriers. In paroxetine-treated females these differences remained statistically significant. In mirtazapine-treated females homozygous for the A-allele compared to AG/GG-carriers, HAMD-17 scores during the study period were constantly and markedly lower, but not statistically different. In males, we found no association between the MAOB A644G intron 13 SNP and antidepressant treatment response. Our data provide first suggestive evidence that the MAOB A644G SNP is involved in the outcome of treatment with mirtazapine or paroxetine in females with major depression. To confirm the role of the MAOB A644G gene variant in antidepressant treatment response, independent replications are needed. If replicated, the MAOB A644G polymorphism could be considered useful for prospective confirmatory pharmacogenetic trials in patients with major depression.     

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Background

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.

Methods

In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.

Results

Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.

Conclusions

Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.     

In vitro effects of D-2-hydroxyglutaric acid on glutamate binding, uptake and release in cerebral cortex of rats

Neurological dysfunction is common in patients with D-2-hydroxyglutaric aciduria (DHGA). However, the mechanisms underlying the neuropathology of this disorder are far from understood. In the present study, we investigated the in vitro effects of D-2-hydroxyglutaric acid (DGA) at various concentrations (0.1-1.0 mM) on various parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomal preparations, Na(+)-dependent L-[3H]glutamate uptake by synaptosomal preparations and Na(+)-independent L-[3H]glutamate uptake by synaptic vesicles, as well as of Na(+)-independent and dependent L-[3H]glutamate binding to synaptic plasma membranes from cerebral cortex of male adult Wistar rats. We observed that DGA significantly increased synaptosomal L-[3H]glutamate uptake, without altering the other parameters. Although these findings do not support a direct excitotoxic action for DGA since the metabolite did not affect important parameters of the main neurotransmission system, they do not exclude a direct action of DGA on NMDA or other glutamate receptors. More comprehensive studies are therefore necessary to evaluate the exact role of DGA on neurotransmission.     

Down-regulation of glutaredoxin by estrogen receptor antagonist renders female mice susceptible to excitatory amino acid mediated complex I inhibition in CNS

beta-N-oxalyl-amino-L-alanine, (L-BOAA), an excitatory amino acid, acts as an agonist of the AMPA subtype of glutamate receptors. It inhibits mitochondrial complex I in motor cortex and lumbosacral cord of male mice through oxidation of critical thiol groups, and glutaredoxin, a thiol disulfide oxido-reductase, helps maintain integrity of complex I. Since incidence of neurolathyrism is less common in women, we examined the mechanisms underlying the gender-related effects. Inhibition of complex I activity by L-BOAA was seen in male but not female mice. Pretreatment of female mice with estrogen receptor antagonist ICI 182,780 or tamoxifen sensitizes them to L-BOAA toxicity, indicating that the neuroprotection is mediated by estrogen receptors. L-BOAA triggers glutathione (GSH) loss in male mice but not in female mice, and only a small but significant increase in oxidized glutathione (GSSG) was seen in females. As a consequence, up-regulation of gamma-glutamyl cysteinyl synthase (the rate-limiting enzyme in glutathione synthesis) was seen only in male mouse CNS but not in females. Both glutathione reductase and glutaredoxin that reduce oxidized glutathione and protein glutathione mixed disulfides, respectively, were constitutively expressed at higher levels in females. Furthermore, glutaredoxin activity in female mice was down-regulated by estrogen antagonist indicating its regulation by estrogen receptor. The higher constitutive expression of glutathione reductase and glutaredoxin could potentially confer neuroprotection to female mice.     

Hyperprolactinaemia in early psychosis—not only due to antipsychotics

Hyperprolactinaemia is often found in patients with schizophrenia and usually considered a consequence of antipsychotics. Prolactin levels were measured in 43 At-Risk Mental State individuals (ARMS) and 26 patients with First Episode Psychosis (FEP). Hyperprolactinaemia was found in 25.6% of ARMS and 46.2% of FEP. Within 60 antipsychotic-naïve ARMS and FEP, hyperprolactinaemia was found in 26.7%. Hyperprolactinaemia may be pre-existing in a subgroup of patients with schizophrenia.     

Impact of 17beta-estradiol on cytokine-mediated apoptotic effects in primary hippocampal and neocortical cell cultures

Estrogens are developmental regulators of mitochondrial apoptotic pathway in the central nervous system, but little is known about their involvement in cytokine-induced apoptosis. In the present study, we evaluated effects of 17beta-estradiol on pro-inflammatory cytokine- and staurosporine-mediated activation of caspase-3 and LDH-release in primary neuronal/glial cell cultures of mouse hippocampal and neocortical cells at different stages of their development in vitro. Enzyme activities were determined with colorimetric methods 6 h, 14 h, 24 h, and 48 h after exposure to the apoptotic agents. Biochemical data were supported at the cellular level by Hoechst 33342 and MAP-2 stainings, which were carried out 48 h after the treatment. Cytokines (co-treatment with Il-1beta and TNFalpha; 1 ng/ml) increased caspase-3 activity in the hippocampal and neocortical cells up to over 200% of control values, and these effects were mostly observed on 2 and 7 days in vitro (DIV). Moderate, but significant cytokine-mediated increase in LDH-release was demonstrated in both tissues, especially on 7 and 12 DIV. Estradiol (100 nM) inhibited the activation of caspase-3 at early stage of development (2 DIV) in the hippocampal, but not in the neocortical cultures. The cytokine-induced activation of caspase-3 and LDH-release was inhibited by estradiol in estrogen receptor-independent way. These data point to a possible role of estrogens as non-estrogen receptor-related inhibitors of cytokine-activated apoptotic pathway in the developing central nervous system.     

Mild surfection of neural cells, especially motoneurons, in primary culture and cell lines

Of all cell types, motoneurons (MNs), are possibly the most difficult to maintain in culture, since their development and survival is conditioned by many factors that are still in the course of identification. This may also be the reason why they are difficult to transfect. We succeed to transfect these fragile cells with lipoplex [DOTAP:PC (10:1)-pGFP]-precoated coverslips. Here, we report that this original method, also termed 'surfection' does not perturbate MN development and survival while giving important transfection yield (15%). Lipofectamine 2000 and other well-known auxiliary lipids (DOPE, Chol) give lower surfection yields. The use of (DOTAP:PC)-based lipid vector also can be extended to several neural and non-neural cell lines with appreciable transfection yield such as a glial cell line (GCL) derived from rat spinal cord (65%), HeLa S3 (60%), COS-7 (30%) and HEK 293 cells (20%). The efficiency of DOTAP:PC (10:1) and Lipofectamine 2000 vectors in our surfection method are compared on standard HeLa S3 cell lines. Lipofectamine 2000 (72%) is slightly better than DOTAP:PC (10:1) (60%). However, the surfection method improved the efficiency of Lipofectamine 2000 itself (72%) as compared to the classical (62%) approach. In summary we have developed an original standard surfection protocol for both MN primary cultures and cell lines, thus simplifying laboratory practice; moreover, Lipofectamine 2000 used in this surfection method is more efficient for the cell lines than the manufacturer-recommended method. We emphasize that our method particularly spares fragile cells like MNs from injure and therefore, might be applied to other fragile cell type in primary cultures.     

Effects of chloride flux modulators in an in vitro model of brain edema formation

Brain edema is a serious consequence of hemispheric stroke and traumatic brain injury and contributes significantly to patient mortality. In the present study, we measured water contents in hippocampal slices as an in vitro model of edema formation. Excitotoxic conditions induced by N-methyl-D-aspartate (NMDA, 300 microM), as well as ischemia induced by oxygen-glucose deprivation (OGD), caused cellular edema formation as indicated by an increase of slice water contents. In the presence of furosemide, an inhibitor of the Na,K,Cl-cotransporter, NMDA-induced edema were reduced by 64% while OGD-induced edema were unaffected. The same observation, i.e., reduction of excitotoxic edema formation but no effect on ischemia-induced edema, was made with chloride transport inhibitors such as DIDS and niflumic acid. Under ischemic conditions, modulation of GABAA receptors by bicuculline, a GABA antagonist, or by diazepam, a GABAergic agonist, did not significantly affect edema formation. Further experiments demonstrated that low chloride conditions prevented NMDA-induced, but not OGD-induced, water influx. Omission of calcium ions had no effect. Our results show that NMDA-induced edema formation is highly dependent on chloride influx as it was prevented by low-chloride conditions and by various compounds that interfere with chloride influx. In contrast, OGD-induced edema observed in brain slices was not affected by modulators of chloride fluxes. The results are discussed with reference to ionic changes occurring during tissue ischemia.     

Biosynthesis of progesterone derived neurosteroids by developing avian CNS : in vitro effects on the gabaa receptor complex

It has been demonstrated in different vertebrate species that the GABA_A receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [[14]C]progesterone metabolites as well as the characterization of their in vitro effects on the GABA_A receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5β-pregnanedione, 5β-pregan-3β-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5β-pregnanedione was converted to 5β-pregnan-3β-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [[3]H]flunitrazepam binding by 5β-pregnan-3β-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5β-pregnan-3β-ol-20-one EC₅₀ values were 0.195±0.049, 0.101±0.017, 0.147±0.009, and 0.569±0.114 μM, and E_max were 22.37±1.57, 23.67±4.02, 29.01±1.08, and 15.11±2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively.In conclusion, the biosynthesis of 5β-pregnan-3β-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABA_A receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.     

Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis

INTRODUCTION: A heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin was isolated from the body of the ascidian Styela plicata (Chordata-Tunicata). The disaccharide composition and the effect of the invertebrate glycan on venous and arterial models of thrombosis was investigated. METHODS AND RESULTS: High performance liquid chromatography of the products formed by a mixture of heparin lyases showed that the ascidian heparin is composed mainly by delta UA(2SO4)-1-->4-beta-d-GlcN(SO4) (47.5%), delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(6SO4) (38.3%) disaccharides and smaller amounts of the disaccharides delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4)(6SO4) (2.8%) and delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4) (8.0%). The invertebrate heparin has an aPTT activity of 18 IU/mg and an antithrombin-mediated antithrombin and anti-factor Xa activities 10-fold lower than that of mammalian heparin. In a venous model of thrombosis in the vena cava, S. plicata heparin inhibits only 80% of thrombosis at a dose 10-fold higher than that of the mammalian heparin that inhibits 100% of thrombosis. However, in an arterio-shunt model of arterial thrombosis, both S. plicata and mammalian heparin possess equivalent antithrombotic activities. It is also shown that at equivalent doses, ascidian heparin has a lower bleeding effect than mammalian heparin. CONCLUSION: The antithrombin-mediated anticoagulant activity of heparin polymers is not directly related to antithrombotic potency in the arterio-venous shunt. The results of the present work suggest that heparin preparations obtained from the body of S. plicata may have a safer therapeutic action in the treatment of arterial thrombosis than mammalian heparin.     

Alterations in nigral NMDA and GABAA receptor control of the striatal dopamine level after repetitive exposures to nitrogen narcosis

Nitrogen pressure exposure in rats results in decreased dopamine (DA) release at the striatal terminals of the substantia nigra pars compacta (SNc) dopaminergic neurons, demonstrating the narcotic potency of nitrogen. This effect is attributed to decreased excitatory and increased inhibitory inputs to dopaminergic neurons, involving a change in NMDA and GABA_A receptor function. We investigated whether repetitive exposures to nitrogen modify the excitatory and inhibitory control of the dopaminergic nigro-striatal pathway.We used voltammetry to measure dopamine levels in freely-moving rats, implanted with dopamine-sensitive electrodes in the striatum. NMDA/GABA_A receptor agonists (NMDA/muscimol) and antagonists (AP7/gabazine) were administered through a guide-cannula into the SNc, and their effects on striatal dopamine levels were measured under normobaric conditions, before and after five repetitive exposures to 1 MPa nitrogen.NMDA-mediated dopamine release was greater following repetitive exposures, AP7-mediated inhibition of glutamatergic input was blocked, suggesting that NMDA receptor sensitivity was increased and glutamate release reduced. Muscimol did not modify dopamine levels following repetitive exposures, whereas the effect of gabazine was greater after exposures than before. This suggested that interneuronal GABA_A receptors were desensitized, leading to an increased GABAergic input at dopaminergic cells. Thus, repetitive nitrogen exposure induced persistent changes in glutamatergic and GABAergic control of dopaminergic neurons, resulting in decreased activity of the nigrostriatal pathway.     

The pentylenetetrazole-induced activity in the hippocampus can be inhibited by the conversion of L-kynurenine to kynurenic acid: an in vitro study

The kynurenine pathway converts tryptophan into various compounds, including l-kynurenine, which in turn can be converted into the excitatory amino acid receptor antagonist kynurenic acid. The ionotropic glutamate receptors have been considered to be attractive targets for new anticonvulsants in neurological disorders such as epileptic seizure. This study was designed to examine the conversion of l-kynurenine to kynurenic acid and to investigate the effects of kynurenic acid on pentylenetetrazole-treated rat brain slices, and in parallel to draw attention to the fact that a well-designed in vitro model has many advantages in pharmacological screening. Schaffer collateral stimulation-evoked field EPSPs were recorded from area CA1 of rat hippocampal slices in vitro; drugs were bath-applied. Pretreatment with the kynurenic acid precursor l-kynurenine led to the elimination of the effect of pentylenetetrazole on hippocampal slices in vitro. N-Omega-nitro-l-arginine, which inhibits kynurenine aminotransferase I and II, abolished this neuroprotective effect.This study has furnished the first in vitro electrophysiological evidence that rat brain slices have the enzymatic capacity to convert exogenously administered l-kynurenine (16 μM) to kynurenic acid in an amount sufficient to protect them against pentylenetetrazole (1 mM)-induced hyperexcitability.     

Low-molecular-weight heparin inhibition in classical complement activaton pathway during pregnancy

Introduction

Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications.

Materials and Methods

The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH₅₀ assay).

Results

The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03 ± 1.20 μg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 µg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG.

Conclusions

We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.     

Significance of NMDA receptor-related glutamatergic amino acid levels in peripheral blood of patients with schizophrenia

Hypo-function of N-methyl d-aspartate (NMDA) receptors is strongly involved in the brain pathophysiology of schizophrenia. Several excitatory amino acids, such as endogenous glutamate, glycine, serine and alanine, which are involved in glutamate neurotransmission via NMDA receptors, were studied to further understand the pathophysiology of schizophrenia and to find a biological marker for this disease, particularly in peripheral blood. In this literature review, we connect several earlier clinical studies and several studies of excitatory amino acid levels in peripheral blood in a historical context. Finally, we join these results and our previous studies, the Juntendo University Schizophrenia Projects (JUSP), which investigated plasma glutamatergic amino acid levels in detail, and considered whether these amino acid levels may be diagnostic, therapeutic, or symptomatic biological markers. This review concludes that peripheral blood levels of endogenous glycine and alanine could be a symptomatic marker in schizophrenia, while peripheral blood levels of exogenous glycine and alanine in augmentation therapies could be therapeutic markers. Noteworthy peripheral blood levels of endogenous d-serine could reflect its brain levels, and may prove to be a useful diagnostic and therapeutic marker in schizophrenia. In addition, measurements of new endogenous molecules, such as glutathione, are promising. Finally, for future therapies with glutamatergic agents still being examined in animal studies, the results of these biological marker studies may lay the foundation for the development of next-generation antipsychotics.     

Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-D-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, beta-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 microM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property.     

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices

During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.     

Metabotropic glutamate receptors modulate synaptic transmission in the perforant path: pharmacology and localization of two distinct receptors

Metabotropic glutamate receptors (mGluRs) have emerged as an interesting family of eight different receptor subtypes that can be divided into three groups according to their pharmacology and sequence similarity. In the present study, the specific mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and

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(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed field excitatory postsynaptic potentials (fEPSPs) in the rat dentate gyrus evoked by perforant path stimulation in a concentration-dependent, rapid and reversible manner (EC₅₀: L-AP4 5.9±1.6 μM, (1S,3R)-ACPD 80±34 μM). In a ‘paired-pulse' stimulation protocol, the first fEPSP showed a stronger reduction, resulting in ‘paired-pulse' facilitation. The effects of L-AP4 but not of (1S,3R)-ACPD could be antagonized by the group III mGluR antagonists (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) and (RS)-α-methyl-4-phosphonophenylglycine (MPPG). Moreover, (1S,3R)-ACPD was still potently depressing fEPSPs after preperfusion of near saturating concentrations of L-AP4. Together, the results suggest that both substances act on different mGluRs. The effects of (1S,3R)-ACPD could not be further differentiated by selective group I or group II mGluR agonists. Although (2S,1′S,2′S)-2-carboxycyclopropylglycine (L-CCG-I) blocked fEPSPs at concentrations 1 μM, these effects, as well as L-AP4 effects, were potently antagonized by MAP4. This suggests that mGluR8 might be responsible for the actions of L-AP4 and L-CCG-I. The two different mGluRs showed a distinct distribution when fEPSPs were recorded simultaneously in the outer and middle molecular layer (OML/MML): The L-AP4 sensitive receptor, possibly mGluR8, seems to be located in the OML while (1S,3R)-ACPD showed its main effect in the MML.     

Cerebral 6-[F]fluoro-l-DOPA uptake in rhesus monkey: pharmacological influence of aromatic amino acid decarboxylase (AAAD) and catechol-O-methyltransferase (COMT) inhibition

FDOPA/PET scans were performed in one rhesus monkey to study the influence of three catechol-O-methyltransferase (COMT) inhibitors (CGP 28014, OR-611 and Ro 40-7592) on FDOPA pharmacokinetics. COMT inhibitors were administered in combination with carbidopa, a peripherally acting inhibitor of the aromatic amino acid decarboxylase (AAAD). FDOPA was administered intravenously and its metabolic fate in plasma was determined using an HPLC system with an on-line γ-γ coincidence detector. Cerebral tracer uptake was assessed in the striatum and in a non-dopaminergic brain region (occipital cortex). In the periphery, the pharmacokinetic efficiency of FDOPA was increased due to the combined inhibition of COMT and AAAD activity. All three COMT inhibitors reduced the FDOPA methylation rate constant in plasma, with complete suppression obtained in the case of Ro 40-7592. In the brain, specific ¹⁸F radioactivity (striatal minus brain reference radioactivity) increased as a result of the increase in FDOPA plasma availability following the administration of COMT and AAAD inhibitors. We established a significant linear correlation between striatal radioactivity and FDOPA plasma levels (r=0.924±0.048, P<0.0001 for total striatal and r=0.948±0.054, P<0.0001 for specific striatal radioactivity). Using plasma FDOPA radioactivity as input, we found that the striatal FDOPA uptake rate constant K_i^FD was not changed by any of the inhibitors. Thus, the enhancement of striatal radioactivity after application of enzyme inhibitors is a consequence of the increase in plasma FDOPA that becomes available for conversion to fluorodopamine in the striatal dopaminergic nerve terminals. By contrast, using the radioactivity in a non-dopaminergic region (cortex) as input, we found that the striatal FDOPA uptake rate constant K_i^ref was significantly (P<0.0001) increased following pretreatment with COMT inhibitors. Our analysis demonstrated that K_i^ref and the 3-OMFD contribution to the cerebral radioactivity were inversely correlated.     

Enhanced release of synaptic glutamate underlies the potentiation of oxygen-glucose deprivation-induced neuronal injury after induction of NOS-2

Reactive nitrogen oxide species (RNOS) may contribute to the progression/enhancement of ischemic injury by augmentation of glutamate release, reduction of glutamate uptake, or a combination of both. Consistent with this, induction of nitric oxide synthase (NOS-2) in murine neocortical cell cultures potentiated neuronal cell death caused by combined oxygen-glucose deprivation in association with a net increase in extracellular glutamate accumulation. However, uptake of glutamate via high affinity, sodium-dependent glutamate transporters was unimpaired by induction of NOS-2 under either aerobic or anaerobic conditions. Further, blocking possible routes of extra-synaptic glutamate release with NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a volume-sensitive organic anion channel blocker, or TBOA (d,l-threo-beta-benzyloxyaspartate), an inhibitor of glutamate transport, exacerbated rather than ameliorated injury. Finally, treatment with riluzole or tetanus toxin attenuated the enhancement in both glutamate accumulation and oxygen-glucose deprivation-induced neuronal injury supporting the idea that increased synaptic release of glutamate underlies, at least in part, the potentiation of neuronal injury by RNOS after NOS-2 induction.     

Neural stem cell grafts reduce the extent of neuronal damage in a mouse model of global ischaemia

The therapeutic potential of neural stem cell transplantation has been well demonstrated in many models of focal brain damage. However, few studies have sought to determine whether neural stem cells are therapeutic in models of diffuse brain injury, such as observed in Alzheimer's disease and global ischaemia. The present study investigated the effects of transplanted MHP36 neural stem cells on the extent of ischaemic damage in a mouse model of global ischaemia and the effects of the immunosuppressive agent cyclosporin A (CsA). C57Bl/6J mice received an intrastriatal graft of MHP36 neural stem cells 3 days after selective neuronal damage had been induced by global ischaemia. The experimental group was subdivided into CsA or saline controls. We discovered that grafts of MHP36 neural stem cells were able to differentiate into neurons and reduce the extent of ischaemic neuronal damage. This reduction was particularly apparent at 4 week post-transplantation and is independent of CsA immunosuppression. MHP36 cells survived robustly in host ischaemic brain and migrated away from the injection tract towards the caudate nucleus and corpus callosum. Although MHP36 grafts were associated with an acute inflammatory response from reactive astrocytes and microglia at 1 week post-transplantation, this decreased markedly by 4 weeks post-transplantation even in the absence of CsA immunosuppression. This is the first study showing a therapeutic benefit of neural stem cells in a highly diffuse brain injury, further highlighting the possibilities of stem cell transplantation for all types of neurodegenerative disease.