In vivo- and in silico-driven identification of novel synthetic quinoxalines as anticonvulsants and AMPA inhibitors.

The lack of effective therapies for epileptic patients and the potentially harmful consequences of untreated seizure incidents have made epileptic disorders in humans a major health concern. Therefore, new and more potent anticonvulsant drugs are continually sought after, to combat epilepsy. On the basis of the pharmacophoric structural specifications of effective α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonists with an efficient anticonvulsant activity, the present work reports the design and synthesis of two novel sets of quinoxaline derivatives. The anticonvulsant activity of the synthesized compounds was evaluated in vivo according to the pentylenetetrazol-induced seizure protocol, and the results were compared with those of perampanel as a reference drug. Among the synthesized compounds, 24, 28, 32, and 33 showed promising activities with ED50 values of 37.50, 23.02, 29.16, and 23.86 mg/kg, respectively. Docking studies of these compounds suggested that AMPA binding could be the mechanism of action of these derivatives. Overall, the pharmacophore-based structural optimization, in vivo and in silico docking, and druglikeness studies indicated that the designed compounds could serve as promising candidates for the development of effective anticonvulsant agents with good pharmacokinetic profiles.

Non-competitive antagonists of NMDA and AMPA receptors decrease seizure-induced c-fos protein expression in the cerebellum and protect against seizure symptoms in adult rats.

The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2 mg/kg), amantadine (50 mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50 mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²âº overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the µ-opioid receptor antagonist CTAP and were not observed in neurons cultured from µ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via µ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of µ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

Behavioral characteristics and pharmacological manipulations of a nicotine-entrainable circadian oscillator.

Chronic daily administration of nicotine and other drugs of abuse has been found to entrain pre- and post-drug circadian locomotor activity episodes that oscillate on a 24-h schedule and persist for several days after administration ceases. This drug-entrainable oscillator system could conceivably lead to circadian rhythms of drug seeking and drug use in human drug addicts. The present study (1) characterizes the ability of daily nicotine administration to entrain circadian wheel-running activity episodes in rats across a range of doses, lighting schedules, and food access; and (2) tests whether pre- and post-nicotine episodes can be altered through pharmacological manipulations. Adult female rats were housed in wheel boxes for 35-60 d, and both wheel-running and feeding-related behaviors were measured continuously. Following acclimation, nicotine or saline was administered for 16-24 d, and the rats were left undisturbed for several test days to observe the persistence of nicotine-entrained activity. The results showed that nicotine dose-dependently entrains wheel-running activity, and the highest dose of 1.0 mg/kg produces robust pre- and post-nicotine circadian activity episodes under constant, fixed, and variable light/dark schedules. In the pharmacological manipulation experiment, nicotine-entrained rats were administered one of seven pharmacological treatments (varenicline, mecamylamine, acamprosate, topiramate, naltrexone, SB-334867, or bupropion) in place of the nicotine injection for 2 d, and the rats were not disturbed for four subsequent days. Most of the treatment drugs significantly reduced post-nicotine activity episodes, but only three treatments affected pre-nicotine episodes: the µ- and κ-opioid receptor antagonist naltrexone, the orexin-1 receptor antagonist SB-334867, and the AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid)/kainate antagonist topiramate. These results show that chronic daily nicotine administration is a robust zeitgeber that dose-dependently entrains a nonphotic oscillator system that includes opioid, orexin, and glutamate pathways.

Glutamatergic input to the lateral hypothalamus stimulates ethanol intake: role of orexin and melanin-concentrating hormone.

BACKGROUND: Glutamate (GLUT) in the lateral hypothalamus (LH) has been suggested to mediate reward behaviors and may promote the ingestion of drugs of abuse. This study tested the hypothesis that GLUT in the LH stimulates consumption of ethanol ( EtOH ) and that this effect occurs, in part, via its interaction with local peptides, hypocretin/orexin (OX), and melanin-concentrating hormone (MCH). METHODS: In Experiments 1 and 2, male Sprague-Dawley rats, after being trained to drink 9% EtOH , were microinjected in the LH with N-methyl-d-aspartate (NMDA) or its antagonist, D-AP5, or with alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) or its antagonist, CNQX-ds. Consumption of EtOH , chow, and water was then measured. To provide an anatomical control, a separate set of rats was injected 2 mm dorsal to the LH. In Experiment 3, the effect of LH injection of NMDA and AMPA on the expression of OX and MCH was measured using radiolabeled in situ hybridization (ISH) and also using digoxigenin-labeled ISH, to distinguish effects on OX and MCH cells in the LH and the nearby perifornical area (PF) and zona incerta (ZI). RESULTS: When injected into the LH, NMDA and AMPA both significantly increased EtOH intake while having no effect on chow or water intake. The GLUT receptor antagonists had the opposite effect, significantly reducing EtOH consumption. No effects were observed with injections 2 mm dorsal to the LH. In addition to these behavioral effects, LH injection of NMDA significantly stimulated expression of OX in both the LH and PF while reducing MCH in the ZI, whereas AMPA increased OX only in the LH and had no effect on MCH. CONCLUSIONS: Glutamatergic inputs to the LH, acting through NMDA and AMPA receptors, appear to have a stimulatory effect on EtOH consumption, mediated in part by increased OX in LH and PF and reduced MCH in ZI.

Maternal care influences hippocampal N-methyl-D-aspartate receptor function and dynamic regulation by corticosterone in adulthood.

BACKGROUND: Variations in maternal care in the rat associate with robust differences in hippocampal development and synaptic plasticity in the offspring. Maternal care also influences pituitary-adrenal stress responses and corticosterone (CORT) regulation of hippocampal plasticity. N-methyl-D-aspartate receptors (NMDAR) regulate synaptic plasticity, and NMDAR function is modulated by stress and CORT. We hypothesized that altered NMDAR function underlies the interaction of maternal and stress effects on hippocampal synaptic plasticity. METHODS: We used electrophysiology and western blot to examine NMDAR synaptic function/expression and NMDAR-dependent long-term potentiation (LTP) in adult offspring of mothers that varied in the frequency of pup licking/grooming (LG) (i.e., High or Low LG). RESULTS: Basal NMDAR synaptic function was enhanced in the hippocampal dentate gyrus (DG) of adult Low LG offspring. Synaptic expression of NMDAR but not α-amino-3-hydroxy-methyl-4-isoxazole propionic acid receptors was also increased. Stress level CORT (100 nmol/L) rapidly (< 20 min) and robustly increased NMDAR function in High LG offspring, eliminating the maternal effect. Corticosterone did not affect NMDAR function in Low LG offspring. Bovine serum albumin-conjugated CORT reproduced the CORT effect in High LG offspring, implicating a membrane-bound corticosteroid receptor. NMDAR hyperfunction might impair synaptic plasticity. Partial NMDAR antagonism by low concentration DL-2-Amino-5-phosphonopentanoic acid rescued a basal LTP deficit in Low LG offspring and inhibited LTP in High LG offspring. CONCLUSIONS: Low LG offspring exhibit basally elevated NMDAR function coupled with insensitivity to CORT modulation indicative of a chronic alteration of NMDAR function. Elevated NMDAR function in the hippocampus might underlie impaired LTP in Low LG offspring.

Origin and properties of striatal local field potential responses to cortical stimulation: temporal regulation by fast inhibitory connections.

Evoked striatal field potentials are seldom used to study corticostriatal communication in vivo because little is known about their origin and significance. Here we show that striatal field responses evoked by stimulating the prelimbic cortex in mice are reduced by more than 90% after infusing the AMPA receptor antagonist CNQX close to the recording electrode. Moreover, the amplitude of local field responses and dPSPs recorded in striatal medium spiny neurons increase in parallel with increasing stimulating current intensity. Finally, the evoked striatal fields show several of the basic known properties of corticostriatal transmission, including paired pulse facilitation and topographical organization. As a case study, we characterized the effect of local GABA(A) receptor blockade on striatal field and multiunitary action potential responses to prelimbic cortex stimulation. Striatal activity was recorded through a 24 channel silicon probe at about 600 µm from a microdialysis probe. Intrastriatal administration of the GABA(A) receptor antagonist bicuculline increased by 65±7% the duration of the evoked field responses. Moreover, the associated action potential responses were markedly enhanced during bicuculline infusion. Bicuculline enhancement took place at all the striatal sites that showed a response to cortical stimulation before drug infusion, but sites showing no field response before bicuculline remained unresponsive during GABA(A) receptor blockade. Thus, the data demonstrate that fast inhibitory connections exert a marked temporal regulation of input-output transformations within spatially delimited striatal networks responding to a cortical input. Overall, we propose that evoked striatal fields may be a useful tool to study corticostriatal synaptic connectivity in relation to behavior.

Neuroprotective effects of mebudipine and dibudipine on cerebral oxygen-glucose deprivation/reperfusion injury.

In the present study, we investigated the effects of mebudipine and dibudipine, two new Ca(2+) channel blockers, on primary murine cortical neurons exposed to oxygen-glucose deprivation/reperfusion. The experiments were performed on cells after 11-16 days of culture. To initiate oxygen-glucose deprivation /reperfusion, the culture medium was replaced by glucose-free medium, and the cells were transferred to a humidified incubation chamber in a mixture of 95% N(2) and 5% CO(2) at 37 degrees C for 30 min. The cultures were pretreated with mebudipine and dibudipine 3 h prior to oxygen-glucose deprivation/reperfusion, in order to explore their effects on neurons under oxygen-glucose deprivation conditions. Cell viability and nitric oxide (NO) production were assessed by MTT assay and the modified Griess method, respectively. Exposure of murine cortical neuronal cells to 30 min oxygen-glucose deprivation significantly decreased cell viability and increased NO production. Pretreatment of the cultures with mebudipine and dibudipine significantly increased cell viability and decreased NO generation in a dose-dependent manner. However, the drugs had no protective effect in cells subjected to oxygen-glucose deprivation for 60 min. Pretreatment of cultures with MK-801 (10 microM), a non-competitive NMDA antagonist, decreased neuronal death after 30-min oxygen-glucose deprivation, while application of NBQX (30 microM), a selective AMPA-kainate receptor antagonist, partially attenuated the cell injury. oxygen-glucose deprivation -induced cytotoxicity and NO production were also inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and MK-801. We conclude that mebudipine and dibudipine could protect cortical neurons against oxygen-glucose deprivation /reperfusion-induced cell injury in a dose-dependent manner, and that this could be mediated partially by decreased NO production.

AMPA antagonists inhibit the extracellular signal regulated kinase pathway and suppress lung cancer growth.

Antagonists at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors limit growth of human cancers in vitro. However, the mechanism of anticancer action of AMPA antagonists is not known. Here we report that the AMPA antagonists GYKI 52466 and CFM-2 inhibit the extracellular signal regulated kinase (ERK1/2) pathway, an intracellular signaling cascade which is activated by growth factors and controls proliferation of lung adenocarcinoma cells. AMPA antagonists reduced phosphorylation of cAMP-responsive element binding protein (CREB), suppressed expression of cyclin D1, upregulated the cell cycle regulators and tumor suppressor proteins p21 and p53 and decreased number of lung adenocarcinoma cells in G2 and S phases of the cell cycle. These findings reveal potential mechanism of antiproliferative action of AMPA antagonists and indicate that this class of compounds may be useful in the therapy of human cancers.

Mild in vitro trauma induces rapid Glur2 endocytosis, robustly augments calcium permeability and enhances susceptibility to secondary excitotoxic insult in cultured Purkinje cells.

Mild brain trauma results in a wide range of neurological symptoms that are not easily explained by the primary pathology. Purkinje neurons of the cerebellum are selectively vulnerable to brain trauma, including indirect remote trauma to the forebrain. This vulnerability manifests itself as a selective and delayed cell loss, for which the underlying mechanisms are poorly understood. Alterations to the surface expression of calcium impermeable AMPA receptors (GluR2-containing) may mediate post-traumatic calcium overload, and initiate biochemical cascades that ultimately cause progressive cell death. Our current study examined this hypothesis using an in vitro model of mild Purkinje trauma, delivered by an elastic stretch at 2.5-2.9 pounds per square inch (psi). This mild trauma alone did not increase cell loss as measured by propidium iodide (PI) uptake (at 20 h) compared to uninjured controls. However, there was a marked increase in cell loss, when cells following mild trauma, were exposed to 10 microM AMPA for 1 h compared to either mild trauma or AMPA exposure alone. Mild injury rendered Purkinje neurons significantly more permeable to AMPA-stimulated (4 microM) calcium influx at 15 min post-injury, including a sustained calcium plateau. This effect was eliminated by inhibiting protein kinase C-dependent GluR2 endocytosis with 2 microM Go6976 or blocking the calcium pore of GluR1/3 containing AMPARs with 500 nM 1-naphthylacetyl spermine (Naspm). Nifedipine (2 microM) eliminated the calcium plateau following mild injury but not the initial spike of Ca2+ increase. These results suggest that mild injuries resulted in a rapid AMPA receptor subtype switch (GluR2 was replaced by GluR1/3), which in turn resulted in an enhanced Ca2+ permeability. We further confirmed this by immunocytochemistry. Dendritic GluR2 co-localization with the pre-synaptic marker synaptophysin was markedly down-regulated at 15 min following mild stretch (P < 0.01), indicative of a rapid decrease in the synaptic expression of receptors containing this subunit. Carboxyfluorescence (CBF) assays revealed that mild stretch did not alter membrane integrity. Finally, we demonstrated that the combination of 500 nM Naspm and 5 nM Go6976 conferred a powerful neuroprotective effect on Purkinje cells by effectively eliminating the effects of mild stretch combined with AMPA in 95% of cells. These results represent a newly described mechanism rendering neurons susceptible to secondary injuries following trauma. Prevention of GluR2 endocytosis may be critical in the development of pharmacotherapies aimed at mild, seemingly inconsequential trauma, to avoid ensuing secondary damage.

Acute effects of AMPA-type glutamate receptor antagonists on intermale social behavior in two mouse lines bidirectionally selected for offensive aggression.

Involvement of AMPA-type glutamate receptors in the regulation of social behavior has been suggested by experiments with mice deficient for the GluR-A subunit-containing AMPA receptors showing reduced intermale aggression. In the present study, effects of AMPA receptor antagonists on mouse social behavior towards unfamiliar Swiss-Webster males on a neutral territory were tested using male subjects from the Turku Aggressive (TA) and Turku Non-Aggressive (TNA) mouse lines bidirectionally selected for high and low levels of offensive aggression. The drugs were the competitive antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), and the non-competitive antagonist 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzenamine (GYKI 52466). In TA mice, CNQX and NBQX decreased the biting component of aggressive structure, while GYKI 52466 suppressed all aggressive manifestations. All drugs increased anxiety-like behavior towards the partner. In TNA mice, NBQX activated mouse social behavior and ambivalent aggression, while CNQX and GYKI 52466 only increased anxiety. Thus, AMPA receptor antagonists affect aggressive behaviors in TA mice supporting the idea that AMPA receptors are involved in the modulation of agonistic impulsive behavioral pattern. GYKI 52466 appeared to be the most selective and efficacious in suppressing the aggression.

In vitro galantamine-memantine co-application: mechanism of beneficial action.

Several drugs are in clinical use for symptomatic treatment of Alzheimer's disease patients. Since Alzheimer's disease is known to be associated with down-regulation of the cholinergic and N-methyl-D-aspartate (NMDA) systems, most of these drugs inhibit acetylcholinesterase, potentiate the activity of nicotinic acetylcholine receptors (nAChRs), or modulate NMDA receptors. Galantamine is an anticholinesterase and allosterically potentiates the activity of the nicotinic receptors. We have recently found that galantamine potentiates the activity of NMDA receptors as well. Memantine is unique in that it inhibits the NMDA receptors. We have developed a hypothesis that combining galantamine and memantine will be more effective for improving the patient's conditions than monotherapy with either drug. Patch clamp and intracellular Ca(2+) imaging experiments using rat cortical and hippocampal neurons clearly provided the in vitro bases for our hypothesis. Memantine blocked the extrasynaptic NMDA receptor 100 times more potently than the synaptic NMDA receptor at negative membrane potentials and the block of both types of NMDA receptors was attenuated with depolarization. However, galantamine potentiation of the NMDA receptors was not voltage dependent. Thus, co-application of memantine with galantamine prevented the galantamine potentiation and the activation of extrasynaptic NMDA receptors, but membrane depolarization revealed the galantamine potentiation. Therefore, cell death is expected to be prevented by memantine near the resting potential while the NMDA-mediated synaptic transmission, which is down-regulated in the patients, is maintained and potentiated by galantamine. These results provide in vitro bases for the beneficial actions of galantamine and memantine combinations.

Contribution of AMPA and NMDA receptors to early and late phases of LTP in hippocampal slices.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor mediated responses were investigated in rat hippocampal slices under 4h of long-term potentiation (LTP) expression. A modified medium containing the NMDA receptor antagonist AP5 and low concentration of Mg(2+) was used to monitor isolated AMPA responses. NMDA components were determined from composite excitatory postsynaptic potentials (EPSPs) under brief (15-20 min) wash-out of AP5. LTP was induced in a medium with low concentration of AP5, resulting in an about two-fold larger increase of the AMPA component than of the NMDA component at both 1h and 4h after induction. Similar results were obtained if LTP was induced in "normal Mg(2+)" and the NMDA components were assessed at the end of experiment, from either composite or isolated NMDA EPSPs, with or without blockade of GABAergic inhibition. It is generally believed that LTP undergoes biochemical and/or structural conversions during the first few hours. Our study, however, shows constant expression of LTP, at least in terms of AMPA versus NMDA components, during this time. The data support the notion that LTP initiates as a predominant amplification of AMPA receptors and remains so for at least 4h.

Structure-activity relationship studies on N3-substituted willardiine derivatives acting as AMPA or kainate receptor antagonists.

N3-substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU(K5)-containing kainate receptors (K(D) 0.105 +/- 0.007 microM vs kainate on native GLU(K5); K(D) 71.4 +/- 8.3 microM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant human GLU(K5), GLU(K5)/GLU(K6), and GLU(K5)/GLU(K2), K(B) values of 0.12 +/- 0.03, 0.12 +/- 0.01, and 0.18 +/- 0.02 microM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU(K6) or GLU(K7) kainate receptors or homomeric GLU(A1-4) AMPA receptors (IC(50) values > 100 microM). Thus, 43 is a potent and selective GLU(K5) receptor antagonist.

Potential metabolites of a condensed 2,3-benzodiazepine derivative.

Putative metabolites of an AMPA antagonist imidazo-2,3-benzodiazepine (2) were synthesized and compared to constituents formed from the parent compound by a rat liver perfusion method. As metabolic transformations, hydroxylation of the 2-methyl group and N-acetylation of the amino functionality in parent compound (2) were registered. The hydroxylated analogue 12 of 2 exhibits a weak AMPA antagonist activity.

Evaluation of interaction between intrathecal adenosine and MK801 or NBQX in a rat formalin pain model.

Adenosine and excitatory amino acids have been known to be involved in modulating nociceptive transmission at the spinal level. The authors assessed the characteristics of the interaction of the adenosine-excitatory amino acid antagonist combinations in the spinal cord of rats on the formalin-induced nociception. Intrathecal NMDA antagonist ((5R, 10S)-(+)-5-methyl-10,11-dihydro-(5)H-dibenzo[a[,]d]cyclohepten-5,10-imine hydrogen maleate, MK801, 30 microg) and AMPA antagonist (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[F]quinoxaline-7-sulfonamide, NBQX, 3 microg) decreased the total number of flinches during both phases in the formalin test. Intrathecal adenosine (300 microg) had little effect on the phase 1 flinching response, but decreased the phase 2 response. The fixed dose analysis and the isobolographic analysis revealed that adenosine interacts additively with MK801 and NBQX in the spinal cord.

17beta-estradiol rescues spinal motoneurons from AMPA-induced toxicity: a role for glial cells.

The ability of astrocytes to mediate 17beta-estradiol neuroprotection of spinal motoneurons challenged with AMPA has been evaluated in a co-culture system in which pure motoneurons were pulsed with 20 microM AMPA and then transferred onto an astrocyte layer pretreated for 24 h with 10 nM 17beta-estradiol. Under these conditions, AMPA toxicity was reverted, an effect that was likely related to increased production and release of GDNF, as shown by RT-PCR, Western blot analysis and ELISA assay. In addition, treatment with GDNF during the 24 h that followed the AMPA pulse produced a similar neuroprotective effect, whereas addition of a neutralizing anti-GDNF antibody prevented neuroprotection. These data suggest a role for astrocytes in the neuroprotective effect of 17beta-estradiol against spinal motoneuron death and find strong support in the marked up-regulation of estrogen receptor alpha found in spinal astrocytes of amyotrophic lateral sclerosis patients.

Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones.

BACKGROUND: The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the alpha-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors. METHODS: The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system. RESULTS: Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC(50) value for AMPA (29 microM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC(50) value for kainate (57 microM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration. CONCLUSIONS: Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

Insulin inhibits AMPA-induced neuronal damage via stimulation of protein kinase B (Akt).

We designed a series of experiments to explore the neuroprotective effects of insulin. Insulin significantly inhibited the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced neuronal cell damage as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. However, insulin had little affect on the AMPA-induced glial cell damage. To determine whether insulin inhibits AMPA-induced excitotoxicity, we performed grease-gap recording assays using rat brain slices. In these experiments, insulin also significantly inhibited AMPA-induced depolarization. Flow cytometry and DNA fragmentation assays showed that insulin inhibits AMPA-induced apoptosis and DNA fragmentation, respectively. Insulin stimulated protein kinase B (Akt) activity, whereas AMPA pretreatment did not alter the insulin-stimulated Akt activity. On the contrary, insulin blocked induction of SAPK/JNK, which AMPA stimulated. Taken together, these results suggest that insulin exerts neuroprotective effects by inhibiting AMPA-induced excitotoxicity and apoptosis, possibly by activating Akt and blocking SAPK/JNK.