Rat Cortical Synaptosomes Research Articles

Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases’ membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

Silymarin Inhibits Glutamate Release and Prevents against Kainic Acid-Induced Excitotoxic Injury in Rats.

Silymarin, a polyphenoic flavonoid derived from the seeds of milk thistle (Silybum marianum), exhibits neuroprotective effects. In this study, we used a model of rat cerebrocortical synaptosomes to investigate whether silymarin affects the release of glutamate, an essential neurotransmitter involved in excitotoxicity. Its possible neuroprotective effect on a rat model of kainic acid (KA)-induced excitotoxicity was also investigated. In rat cortical synaptosomes, silymarin reduced glutamate release and calcium elevation evoked by the K+ channel blocker 4-aminopyridine but did not affect glutamate release caused by the Na+ channel activator veratridine or the synaptosomal membrane potential. Decreased glutamate release by silymarin was prevented by removal of extracellular calcium and blocking of N- and P/Q-type Ca2+ channel or extracellular signal-regulated kinase 1/2 (ERK1/2) but not by blocking of intracellular Ca2+ release. Immunoblotting assay results revealed that silymarin reduced 4-aminopyridine-induced phosphorylation of ERK1/2. Moreover, systemic treatment of rats with silymarin (50 or 100 mg/kg) 30 min before systemic KA (15 mg/kg) administration attenuated KA-induced seizures, glutamate concentration elevation, neuronal damage, glial activation, and heat shock protein 70 expression as well as upregulated KA-induced decrease in Akt phosphorylation in the rat hippocampus. Taken together, the present study demonstrated that silymarin depressed synaptosomal glutamate release by suppressing voltage-dependent Ca2+ entry and ERK1/2 activity and effectively prevented KA-induced in vivo excitotoxicity.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4β2, α4β4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

ATR-IR and EPR spectroscopy for following the membrane restoration of isolated cortical synaptosomes in aluminium-induced Alzheimer’s disease – Like rat model

Synaptosomal membrane peroxidation and alteration in its biophysical properties are associated with Aluminium (Al) toxicity that may lead to cognitive dysfunction and Alzheimer’s disease (AD) like pathogenesis. Here we investigated the therapeutic potential of Lepedium sativum (LS) as a natural anti-inflammatory, antioxidant and as acetyl cholinesterase inhibitor in treating Al induced AD-like in rat model. We utilized ATR-IR spectroscopy to follow the restoration in the damaged membrane structure of isolated rat cortical synaptosomes and its biophysical properties, electron paramagnetic resonance (EPR) spin trapping to follow NADPH oxidase activity (NOX), and EPR spin labelling in response to LS treatment after Al intoxication. We measured the concentration of Ca2+ ions in rat cortical tissue by inductively coupled plasma (ICP), the brain atrophy/curing and hydrocephalus by magnetic resonance imaging (MRI) besides light microscope histopathology. Our results revealed significant increase in synaptosomal membrane rgidification, order, lipid packing, reactive oxygen species (ROS) production and Ca2+ ion concentration as a result of Al intoxication. The dramatic increase in Ca2+ ion concentration detected in AD group associated with the increase in synaptic membrane polarity and EPR-detected order S-parameter suggest that release of synaptic vesicles into synaptic cleft might be hindered. LS treatment reversed these changes in synaptic membranes, and rescued an observed deficit in the exploratory behaviour of AD group. Our results also strongly suggest that the synaptosomal membrane phospholipids that underwent free radical attacks mediated by AlCl3, due to greater NOX activity, was prevented in the LS group. The results of ATR-IR and EPR spectroscopic techniques recommend LS as a promising therapeutic agent against synaptic membrane alterations opening a new window for AD drug developers.

ATR-IR and EPR spectroscopy for detecting the alterations in cortical synaptosomes induced by aluminium stress.

Aluminium (Al) is reported to promote free radical production, decrease the antioxidant enzyme status and disturb the enzyme activity involved in acetylcholine metabolism leading to cognitive dysfunction that are strongly associated with Alzheimer's disease (AD) pathogenesis. This work aimed at investigating the effect of Al-toxicity on synaptosomal membrane biophysical properties and lipid peroxidation during 65 days. We utilized ATR-IR spectroscopy to study the changes in membrane biochemical structure and biophysical properties of isolated rat cortical synaptosomes, and EPR spin trapping and labeling to follow NADPH oxidase activity and changes of membrane order parameter, respectively. The results showed increase in membrane fluidity and disorder in early 21d of AlCl3 treatment, while after 42d the membrane rigidity, packing, and order increased. The late (65d) an increase in the amount of unsaturated fatty acids, the accumulation of lipid peroxide end products, and ROS production were detected in rat cortex synaptosomes mediated by Al toxicity and oxidative stress (OS). A dramatic increase was also detected in Ca2+ level, synaptic membrane polarity, and EPR-detected order S-parameter. These outcomes strongly suggest that the synaptosomal membrane phospholipids underwent free radical attacks mediated by AlCl3 due to greater NOX activity, and the release of synaptic vesicles into synaptic cleft might be hindered. The adopted spectroscopic techniques have shed light on the biomolecular structure and membrane biophysical changes of isolated cortical synaptosomes for the first time, allowing researchers to move closer to a complete understanding of pathological tissues.

Abstract WP337: The Ischemic Neuroprotectant Protein Kinase C Epsilon, Phosphorylates Enzymes in Neuronal NADH Shuttle and Regulates Mitochondrial Respiration and Glycolysis

Background: Among PKCε-mediated potential protective mechanisms in ischemic preconditioning, one target that we surmise is crucial for neuroprotection is the malate-aspartate shuttle (MAS). MAS is the most important NAD + /NADH shuttle importing reducing equivalents (NADH) generated by glycolysis from the cytosol into the mitochondria, and thus, tightly controls neuronal respiration. We hypothesized that PKCε regulates MAS in the brain and mediates neuronal mitochondrial respiration. Method: Phosphoproteomics analysis and immunoprecipitation (IP) were used to determine PKCε-induced phosphorylation of MAS components. Synaptosomes from cortices of naïve SD rats were treated with Tat-conjugated ΨεRACK (selective PKCε activator) or control Tat peptide and sent for phosphoproteomics analysis. To confirm the predicted phosphorylation, IP was performed. SD rats were treated with ΨεRACK (i.p.) or Tat and sacrificed 48hrs later. Proteins from rat cortical synaptosomes were immunoprecipitated with phosphoserine/threonine/tyrosine and immunoblotted for predicted phosphorylated proteins. Once phosphorylation was determined, we evaluated energy metabolism in primary neurons. Neuronal cultures were obtained from embryonic pups cortices and treated with ΨεRACK or Tat on DIV 7-10. Cellular respiration and glycolytic function were evaluated 48hrs after treatment using Seahorse XF Analyzer. Result: Phosphoproteomics analysis and IP results revealed increased phosphorylation of key MAS components: the mitochondrial aspartate aminotransferase (GOT2) and the oxoglutarate carrier (OGC). After PKCε activation, GOT2 and OGC exhibited serine phosphorylation increased by 2.14 fold (P < 0.05, n=4-5), and 1.74 fold (P < 0.01, n=4-5), respectively. In addition, in primary neurons, ΨεRACK treatment increased the basal and maximum cellular respiration by 0.13 (P < 0.05, n=4), and 0.22 fold (P < 0.01, n=4), respectively. The basal and maximum glycolysis were increased by 0.14, and 0.21 fold (P<0.05, n=4), respectively. Conclusion: Our data strongly suggest that PKCε regulates MAS, and increases neuronal metabolism by promoting cellular respiration and glycolysis. These changes may be linked to our observed PKCε-induced ischemic tolerance in the brain.

PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria nigriventer spider venom, releases l-glutamate from rat brain synaptosomes involving Na+ and Ca2+ channels and changes protein expression at the blood-brain barrier

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally.

Lycopene depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase C in rat cerebrocortical nerve terminals.

Lycopene is a natural dietary carotenoid that was reported to exhibit a neuroprotective profile. Considering that excitotoxicity and cell death induced by glutamate are involved in many brain disorders, the effect of lycopene on glutamate release in rat cerebrocortical nerve terminals and the possible mechanism involved in such effect was investigated. We observed here that lycopene inhibited 4-aminopyridine (4-AP)-evoked glutamate release and intrasynaptosomal Ca2+ concentration elevation. The inhibitory effect of lycopene on 4-AP-evoked glutamate release was markedly reduced in the presence of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was insensitive to the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Furthermore, in the presence of the protein kinase C inhibitors GF109203X and Go6976, the action of lycopene on evoked glutamate release was prevented. These results are the first to suggest that lycopene inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic Ca2+ entry and protein kinase C activity.

RETRACTED ARTICLE: Increased Aβ42-α7-like nicotinic acetylcholine receptor complex level in lymphocytes is associated with apolipoprotein E4-driven Alzheimer’s disease pathogenesis

BackgroundThe apolipoprotein E ε4 (APOE4) genotype is a prominent late-onset Alzheimer’s disease (AD) risk factor. ApoE4 disrupts memory function in rodents and may contribute to both plaque and tangle formation.MethodsCoimmunoprecipitation and Western blot detection were used to determine: 1) the effects of select fragments from the apoE low-density lipoprotein (LDL) binding domain and recombinant apoE subtypes on amyloid beta (Aβ)42-α7 nicotinic acetylcholine receptor (α7nAChR) interaction and tau phosphorylation in rodent brain synaptosomes; and 2) the level of Aβ42-α7nAChR complexes in matched controls and patients with mild cognitive impairment (MCI) and dementia due to AD with known APOE genotypes.ResultsIn an ex vivo study using rodent synaptosomes, apoE141–148 of the apoE promotes Aβ42-α7nAChR association and Aβ42-induced α7nAChR-dependent tau phosphorylation. In a single-blind study, we examined lymphocytes isolated from control subjects, patients with MCI and dementia due to AD with known APOE genotypes, sampled at two time points (1 year apart). APOE ε4 genotype was closely correlated with heightened Aβ42-α7nAChR complex levels and with blunted exogenous Aβ42 effects in lymphocytes derived from AD and MCI due to AD cases. Similarly, plasma from APOE ε4 carriers enhanced the Aβ42-induced Aβ42-α7nAChR association in rat cortical synaptosomes. The progression of cognitive decline in APOE ε4 carriers correlated with higher levels of Aβ42-α7nAChR complexes in lymphocytes and greater enhancement by their plasma of Aβ42-induced Aβ42-α7nAChR association in rat cortical synaptosomes.ConclusionsOur data suggest that increased lymphocyte Aβ42-α7nAChR-like complexes may indicate the presence of AD pathology especially in APOE ε4 carriers. We show that apoE, especially apoE4, promotes Aβ42-α7nAChR interaction and Aβ42-induced α7nAChR-dependent tau phosphorylation via its apoE141–148 domain. These apoE-mediated effects may contribute to the APOE ε4-driven neurodysfunction and AD pathologies.

Inhibition of glutamate release by cilnidipine in rat cerebrocortical nerve terminals (synaptosomes).

Cilnidipine is an antihypertensive drug that was reported to have a neuroprotective profile. The present study aimed to investigate the effect of cilnidipine on the 4-aminopyridine (4-AP)-induced glutamate release in the rat cerebral cortex using isolated nerve terminals (synaptosomes). Cilnidipine reduced the release of glutamate release induced by 4-AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the 4-AP-induced intrasynaptosomal Ca concentration elevation and was not because of an alteration of the synaptosomal membrane potential. The inhibition of glutamate release by cilnidipine was markedly reduced or eliminated in the presence of the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC and the protein kinase A inhibitor H89. The intracellular Ca-release inhibitors dantrolene and CGP37157, the mitogen-activated protein kinase inhibitor PD98059 or the protein kinase C inhibitor GF109203X failed to affect the action of cilnidipine. These results suggest that cilnidipine inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca entry and protein kinase A activity.

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices

Context: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals.Objective: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats.Materials and methods: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 μM).Results: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 μM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes.Discussion and conclusion: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.

P2X7 receptor activation downmodulates Na+-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na+-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na+ influx across plasma membrane. 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP, 3–300μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [3H]GABA (14%) and [14C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3μM), prevented inhibition of [3H]GABA and [14C]glutamate uptake by BzATP (100μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na+ and was mimicked by Na+ ionophores, like gramicidin and monensin. Increases in intracellular Na+ (with veratridine or ouabain) or substitution of extracellular Na+ by N-methyl-d-glucamine (NMDG)+ all decreased [3H]GABA and [14C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100μM) was also attenuated by calmidazolium, which selectively inhibits Na+ currents through the P2X7 receptor pore. In conclusion, disruption of the Na+ gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

Dimebon, an antihistamine drug, inhibits glutamate release in rat cerebrocortical nerve terminals

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of dimebon, an antihistamine with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. Dimebon inhibited the release of glutamate that was evoked by exposing the synaptosomes to the K+ channel blocker 4-aminopyridine, and this effect was prevented by chelating extracellular Ca2+ ions, and the vesicular transporter inhibitor bafilomycin A1. Dimebon inhibited depolarization-evoked increase in cytosolic free Ca2+ concentration, and the dimebon-mediated inhibition of glutamate release was prevented by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC. The inhibitory action of dimebon on glutamate release was not due to its decreasing synaptosomal excitability, because dimebon did not alter the resting synaptosomal membrane potential or 4-aminopyridine-mediated depolarization. Furthemore, the dimebon effect on 4-aminopyridine-evoked glutamate release was prevented by the protein kinase C inhibitor, and dimebon substantially reduced the 4-AP-induced phosphorylation of protein kinase C. However, the dimebon-mediated inhibition of glutamate release was unaffected by the N-methyl-d-aspartate receptor agonist or antagonist. These results suggest that dimebon inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic voltage-dependent Ca2+ entry and protein kinase C activity. This implies that the inhibition of glutamate release is an additional pharmacological activity of dimebon that may play a critical role in the apparent clinical efficacy of this compound.

Antidepressant-like activity of YL-0919: a novel combined selective serotonin reuptake inhibitor and 5-HT1A receptor agonist.

It has been suggested that drugs combining activities of selective serotonin reuptake inhibitor and 5-HT1A receptor agonist may form a novel strategy for higher therapeutic efficacy of antidepressant. The present study aimed to examine the pharmacology of YL-0919, a novel synthetic compound with combined high affinity and selectivity for serotonin transporter and 5-HT1A receptors. We performed in vitro binding and function assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of YL-0919. YL-0919 displayed high affinity in vitro to both 5-HT1A receptor and 5-HT transporter prepared from rat cortical tissue. It exerted an inhibitory effect on forskolin-stimulated cAMP formation and potently inhibited 5-HT uptake in both rat cortical synaptosomes and recombinant cells. After acute p.o. administration, very low doses of YL-0919 reduced the immobility time in tail suspension test and forced swimming test in mice and rats, with no significant effect on locomotor activity in open field test. Furthermore, WAY-100635 (a selective 5-HT1A receptor antagonist, 0.3 mg/kg) significantly blocked the effect of YL-0919 in tail suspension test and forced swimming test. In addition, chronic YL-0919 treatment significantly reversed the depressive-like behaviors in chronically stressed rats. These findings suggest that YL-0919, a novel structure compound, exerts dual effect on the serotonergic system, as both 5-HT1A receptor agonist and 5-HT uptake blocker, showing remarkable antidepressant effects in animal models. Therefore, YL-0919 may be used as a new option for the treatment of major depressive disorder.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Quercetin inhibits depolarization-evoked glutamate release in nerve terminals from rat cerebral cortex

Quercetin, a naturally occurring flavonoid, has been reported to have a neuroprotective profile. An excessive release of glutamate is widely considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. This study investigated whether quercetin affected glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Quercetin inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP), and this effect was prevented by the chelating extracellular Ca2+ ions. Quercetin decreased the depolarization-induced increase in the cytosolic free Ca2+ concentration ([Ca2+]C), whereas it did not alter 4-AP-mediated depolarization and Na+ influx. The quercetin-mediated inhibition of glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca2+ release. Combined inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the inhibitory effect of quercetin on evoked glutamate release. Furthermore, quercetin decreased the 4-AP-induced phosphorylation of PKC and PKA. These results suggest that quercetin inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in presynaptic voltage-dependent Ca2+ entry and to the suppression of PKC and PKA activity.

Antidepressant-like effects of 071031B, a novel serotonin and norepinephrine reuptake inhibitor

SNRIs (serotonin and norepinephrine reuptake inhibitors) have been proposed to exert increased therapeutic efficacy or be faster acting compared to commonly used antidepressants. In this study, we performed in vitro binding and uptake assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of the compound 071031B; we also performed cytotoxicity tests using HepG2 cells and SH-SY5Y cells to predict the toxicity of 071031B. In vitro, 071031B had high affinity for both serotonin transporters and norepinephrine transporters prepared from rat cortex tissue (Ki=2.68 and 1.09nM, respectively) and recombinant cells (Ki=1.57 and 0.36nM, respectively). Moreover, 071031B also potently inhibited the uptake of serotonin (5-HT) and norepinephrine (NE) into rat cortical synaptosomes (Ki=1.99 and 1.09nM, respectively) and recombinant cells (Ki=3.23 and 0.79nM, respectively). In vivo, acute administration of 071031B dose-dependently reduced the immobility time in the tail suspension test in mice and the forced swimming test in mice and rats with higher efficacy than duloxetine and showed no stimulatory effect on the locomotor activity. Chronic 071031B treatment (5 or 10mg/kg) significantly reversed depressive-like behaviors in chronically stressed rats, including reduced sucrose preference, decreased locomotor activity, and prolonged latency to begin eating. Furthermore, 071031B also exhibited lower cytotoxicity in HepG2 cells and SH-SY5Y cells in vitro than duloxetine. These findings suggest that 071031B is a novel, balanced serotonin and norepinephrine reuptake inhibitor, with more potent antidepressant effects and lower hepatotoxicity and neurotoxicity in vitro than duloxetine.

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K+ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca2+ ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca2+]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na+/Ca2+ exchanger blocker CGP37157. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca2+ entry and PKC activity.