4-phenylselenyl-7-chloroquinoline, a novel multitarget compound with anxiolytic activity: Contribution of the glutamatergic system.

A growing body of evidence demonstrates that quinoline compounds have attracted much attention in the field of drug development. Accordingly, 4-phenylselenyl-7-chloroquinoline (4-PSQ) is a new quinoline derivative containing selenium, which showed a potential antioxidant, antinociceptive and anti-inflammatory effect. The present study was undertaken to evaluate the anxiolytic-like properties of 4-PSQ. Mice were orally pretreated with 4-PSQ (5-50 mg/kg) or vehicle, 30 min prior to the elevated plus-maze (EPM), light-dark (LDT) or open field (OFT) tests. A time-response curve was carried out by administration of 4-PSQ (50 mg/kg) at different times before the EPM test. The involvement of glutamate uptake/release and Na+, K+-ATPase activity in the anxiolytic-like effect was investigated in cerebral cortices. In addition, the effectiveness of acute treatment with 4-PSQ was evaluated in a model of kainate (KA)-induced anxiety-related behavior. Finally, acute toxicity of this compound was investigated. 4-PSQ produced an anxiolytic-like action, both in EPM and LDT. In OFT, 4-PSQ did not affect locomotor and exploratory activities. 4-PSQ anxiolytic-like effect started at 0.5 h and remained significant up to 72 h after administration. Treatment with 4-PSQ reduced [3H] glutamate uptake, but the [3H] glutamate release and Na+, K+-ATPase activity were not altered. KA-induced anxiety-related behavior was protected by 4-PSQ pretreatment. Additionally, 4-PSQ exposure did not alter urea levels, aspartate (AST) and alanine aminotrasferase (ALT) activities in plasma. Parameters of oxidative stress in brain and liver of mice were not modified by 4-PSQ. Taken together these data demonstrated that the anxiolytic-like effect caused by 4-PSQ seems to be mediated by involvement of the glutamatergic system.

Melatonin reduces excitotoxic blood-brain barrier breakdown in neonatal rats.

The blood-brain barrier (BBB) is a complex structure that protects the central nervous system from peripheral insults. Understanding the molecular basis of BBB function and dysfunction holds significant potential for future strategies to prevent and treat neurological damage. The aim of our study was (1) to investigate BBB alterations following excitotoxicity and (2) to test the protective properties of melatonin. Ibotenate, a glutamate analog, was injected intracerebrally in postnatal day 5 (P5) rat pups to mimic excitotoxic injury. Animals were than randomly divided into two groups, one receiving intraperitoneal (i.p.) melatonin injections (5mg/kg), and the other phosphate buffer saline (PBS) injections. Pups were sacrificed 2, 4 and 18 h after ibotenate injection. We determined lesion size at 5 days by histology, the location and organization of tight junction (TJ) proteins by immunohistochemical studies, and BBB leakage by dextran extravasation. Expression levels of BBB genes (TJs, efflux transporters and detoxification enzymes) were determined in the cortex and choroid plexus by quantitative PCR. Dextran extravasation was seen 2h after the insult, suggesting a rapid BBB breakdown that was resolved by 4h. Extravasation was significantly reduced in melatonin-treated pups. Gene expression and immunohistochemical assays showed dynamic BBB modifications during the first 4h, partially prevented by melatonin. Lesion-size measurements confirmed white matter neuroprotection by melatonin. Our study is the first to evaluate BBB structure and function at a very early time point following excitotoxicity in neonates. Melatonin neuroprotects by preventing TJ modifications and BBB disruption at this early phase, before its previously demonstrated anti-inflammatory, antioxidant and axonal regrowth-promoting effects.

Melanocortin receptor agonist ACTH 1-39 protects rat forebrain neurons from apoptotic, excitotoxic and inflammation-related damage.

Patients with relapsing-remitting multiple sclerosis (RRMS) are commonly treated with high doses of intravenous corticosteroids (CS). ACTH 1-39, a member of the melanocortin family, stimulates production of CS by the adrenals, but melanocortin receptors are also found in the central nervous system (CNS) and on immune cells. ACTH is produced within the CNS and may have direct protective effects on glia and neurons independent of CS. We previously reported that ACTH 1-39 protected oligodendroglia (OL) and their progenitors (OPC) from a panel of excitotoxic and inflammation-related agents. Neurons are the most vulnerable cells in the CNS. They are terminally differentiated, and sensitive to inflammatory and excitotoxic insults. For potential therapeutic protection of gray matter, it is important to investigate the direct effects of ACTH on neurons. Cultures highly enriched in neurons were isolated from 2-3 day old rat brain. After 4-7 days in culture, the neurons were treated for 24h with selected toxic agents with or without ACTH 1-39. ACTH 1-39 protected neurons from death induced by staurosporine, glutamate, NMDA, AMPA, kainate, quinolinic acid, reactive oxygen species and, to a modest extent, from rapidly released NO, but did not protect against kynurenic acid or slowly released nitric oxide. Our results show that ACTH 1-39 protects neurons in vitro from several apoptotic, excitotoxic and inflammation-related insults.

Cannabinoid receptor agonists reduce the short-term mitochondrial dysfunction and oxidative stress linked to excitotoxicity in the rat brain.

The endocannabinoid system (ECS) is involved in a considerable number of physiological processes in the Central Nervous System. Recently, a modulatory role of cannabinoid receptors (CBr) and CBr agonists on the reduction of the N-methyl-d-aspartate receptor (NMDAr) activation has been demonstrated. Quinolinic acid (QUIN), an endogenous analog of glutamate and excitotoxic metabolite produced in the kynurenine pathway (KP), selectively activates NMDAr and has been shown to participate in different neurodegenerative disorders. Since the early pattern of toxicity exerted by this metabolite is relevant to explain the extent of damage that it can produce in the brain, in this work we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) and other agonists (anandamide or AEA, and CP 55,940 or CP) on early markers of QUIN-induced toxicity in rat striatal cultured cells and rat brain synaptosomes. WIN, AEA and CP exerted protective effects on the QUIN-induced loss of cell viability. WIN also preserved the immunofluorescent signals for neurons and CBr labeling that were decreased by QUIN. The QUIN-induced early mitochondrial dysfunction, lipid peroxidation and reactive oxygen species (ROS) formation were also partially or completely prevented by WIN pretreatment, but not when this CBr agonist was added simultaneously with QUIN to brain synaptosomes. These findings support a neuroprotective and modulatory role of cannabinoids in the early toxic events elicited by agents inducing excitotoxic processes.

Excessive disgust caused by brain lesions or temporary inactivations: mapping hotspots of the nucleus accumbens and ventral pallidum.

Disgust is a prototypical type of negative affect. In animal models of excessive disgust, only a few brain sites are known in which localized dysfunction (lesions or neural inactivations) can induce intense 'disgust reactions' (e.g. gapes) to a normally pleasant sensation such as sweetness. Here, we aimed to map forebrain candidates more precisely, to identify where either local neuronal damage (excitotoxin lesions) or local pharmacological inactivation (muscimol/baclofen microinjections) caused rats to show excessive sensory disgust reactions to sucrose. Our study compared subregions of the nucleus accumbens shell, ventral pallidum, lateral hypothalamus, and adjacent extended amygdala. The results indicated that the posterior half of the ventral pallidum was the only forebrain site where intense sensory disgust gapes in response to sucrose were induced by both lesions and temporary inactivations (this site was previously identified as a hedonic hotspot for enhancements of sweetness 'liking'). By comparison, for the nucleus accumbens, temporary GABA inactivations in the caudal half of the medial shell also generated sensory disgust, but lesions never did at any site. Furthermore, even inactivations failed to induce disgust in the rostral half of the accumbens shell (which also contains a hedonic hotspot). In other structures, neither lesions nor inactivations induced disgust as long as the posterior ventral pallidum remained spared. We conclude that the posterior ventral pallidum is an especially crucial hotspot for producing excessive sensory disgust by local pharmacological/lesion dysfunction. By comparison, the nucleus accumbens appears to segregate sites for pharmacological disgust induction and hedonic enhancement into separate posterior and rostral halves of the medial shell.

Mechanism based neurotoxicity of mGlu5 positive allosteric modulators--development challenges for a promising novel antipsychotic target.

Previous work has suggested that activation of mGlu5 receptor augments NMDA receptor function and thereby may constitute a rational approach addressing glutamate hypofunction in schizophrenia and a target for novel antipsychotic drug development. Here, we report the in vitro activity, in vivo efficacy and safety profile of 5PAM523 (4-Fluorophenyl){(2R,5S)-5-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]-2-methylpiperidin-1-yl}methanone), a structurally novel positive allosteric modulator selective of mGlu5. In cells expressing human mGlu5 receptor, 5PAM523 potentiated threshold responses to glutamate in fluorometric calcium assays, but does not have any intrinsic agonist activity. 5PAM523 acts as an allosteric modulator as suggested by the binding studies showing that 5PAM523 did not displace the binding of the orthosteric ligand quisqualic acid, but did partially compete with the negative allosteric modulator, MPyEP. In vivo, 5PAM523 reversed amphetamine-induced locomotor activity in rats. Therefore, both the in vitro and in vivo data demonstrate that 5PAM523 acts as a selective mGlu5 PAM and exhibits anti-psychotic like activity. To study the potential for adverse effects and particularly neurotoxicity, brain histopathological exams were performed in rats treated for 4 days with 5PAM523 or vehicle. The brain exam revealed moderate to severe neuronal necrosis in the rats treated with the doses of 30 and 50 mg/kg, particularly in the auditory cortex and hippocampus. To investigate whether this neurotoxicity is mechanism specific to 5PAM523, similar safety studies were carried out with three other structurally distinct selective mGlu5 PAMs. Results revealed a comparable pattern of neuronal cell death. Finally, 5PAM523 was tested in mGlu5 knock-out (KO) and wild type (WT) mice. mGlu5 WT mice treated with 5PAM523 for 4 days at 100 mg/kg presented significant neuronal death in the auditory cortex and hippocampus. Conversely, mGlu5 KO mice did not show any neuronal loss by histopathology, suggesting that enhancement of mGlu5 function is responsible for the toxicity of 5PAM523. This study reveals for the first time that augmentation of mGlu5 function with selective allosteric modulators results in neurotoxicity.

Attack of the nervous system by Clostridium perfringens Epsilon toxin: from disease to mode of action on neural cells.

Epsilon toxin (ET), produced by Clostridium perfringens types B and D, ranks among the four most potent poisonous substances known so far. ET-intoxication is responsible for enterotoxaemia in animals, mainly sheep and goats. This disease comprises several manifestations indicating the attack of the nervous system. This review aims to summarize the effects of ET on central nervous system. ET binds to endothelial cells of brain capillary vessels before passing through the blood-brain barrier. Therefore, it induces perivascular oedema and accumulates into brain. ET binding to different brain structures and to different component in the brain indicates regional susceptibility to the toxin. Histological examination has revealed nerve tissue and cellular lesions, which may be directly or indirectly caused by ET. The naturally occurring disease caused by ET-intoxication can be reproduced experimentally in rodents. In mice and rats, ET recognizes receptor at the surface of different neural cell types, including certain neurons (e.g. the granule cells in cerebellum) as well as oligodendrocytes, which are the glial cells responsible for the axons myelination. Moreover, ET induces release of glutamate and other transmitters, leading to firing of neural network. The precise mode of action of ET on neural cells remains to be determined.

Tardive dyskinesia is caused by maladaptive synaptic plasticity: a hypothesis.

It has been 50 years since the first patients with tardive dyskinesia (TD) were described, but the pathophysiology is only partially understood and effective treatments have remained elusive. Newer atypical antipsychotics with less nonspecific activity at dopamine receptors have not heralded the end of tardive dyskinesia and merely highlight the incomplete understanding of the disorder. We present an overview of the existing pathophysiology of the disorder and incorporate recent developments in genetics and the study of human synaptic plasticity in other hyperkinetic movement disorders. We propose a hypothesis that dopamine-receptor sensitization and altered function of the N-methyl-D-aspartate receptor produces maladaptive synaptic plasticity, which allows the encoding of abnormal motor programs, and propose studies that would falsify or support this hypothesis. In conclusion, a maladaptive synaptic plasticity" hypothesis goes some way toward filling in the gaps of existing theories of TD with the pathophysiology of other hyperkinetic movement disorders. © 2012 Movement Disorder Society.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A(2B) receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A(2B) receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

Neuroprotective potential of biphalin, multireceptor opioid peptide, against excitotoxic injury in hippocampal organotypic culture.

UNLABELLED: Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. FINDINGS: (1) 0.025-0.1 µM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.

Respiratory motoneurons and pathological conditions: lessons from hypoglossal motoneurons challenged by excitotoxic or oxidative stress.

Hypoglossal motoneurons (HMs) are respiration-related brainstem neurons that command rhythmic contraction of the tongue muscles in concert with the respiratory drive. In experimental conditions, HMs can exhibit a range of rhythmic patterns that may subserve different motor outputs and functions. Neurodegenerative diseases like amyotrophic lateral sclerosis (ALS; Lou-Gehrig disease) often damage HMs with distressing symptoms like dysarthria, dysphagia and breathing difficulty related to degeneration of respiratory motoneurons. While the cause of ALS remains unclear, early diagnosis remains an important goal for potential treatment because fully blown clinical symptoms appear with degeneration of about 30% motoneurons. Using a simple in vitro model of the rat brainstem to study the consequences of excitotoxicity or oxidative stress (believed to occur during the onset of ALS) on HMs, it is possible to observe distinct electrophysiological effects associated with HM experimental pathology. In fact, excitotoxicity caused by glutamate uptake block triggers sustained bursting and enhanced synaptic transmission, whereas oxidative stress generates slow depolarization, augmented repeated firing, and decreased synaptic transmission. In either case, only a subpopulation of HMs shows abnormal functional changes. Although these two insults induce separate functional signatures, the consequences on HMs after a few hours are similar and are preceded by activation of the stress transcription factor ATF-3. The deleterious action of excitotoxicity is inhibited by early administration of riluzole, a drug currently employed for the symptomatic treatment of ALS, demonstrating that this in vitro model can be useful for testing potential neuroprotective agents.

Microsomal prostaglandin E synthase-1 contributes to ischaemic excitotoxicity through prostaglandin E2 EP3 receptors.

BACKGROUND AND PURPOSE: Although microsomal prostaglandin E synthase (mPGES)-1 is known to contribute to stroke injury, the underlying mechanisms remain poorly understood. This study examines the hypothesis that EP(3) receptors contribute to stroke injury as downstream effectors of mPGES-1 neurotoxicity through Rho kinase activation. EXPERIMENTAL APPROACH: We used a glutamate-induced excitotoxicity model in cultured rat and mouse hippocampal slices and a mouse middle cerebral artery occlusion-reperfusion model. Effects of an EP(3) receptor antagonist on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice. KEY RESULTS: In cultures of rat hippocampal slices, the mRNAs of EP(1-4) receptors were constitutively expressed and only the EP(3) receptor antagonist ONO-AE3-240 attenuated and only the EP(3) receptor agonist ONO-AE-248 augmented glutamate-induced excitotoxicity in CA1 neurons. Hippocampal slices from mPGES-1 KO mice showed less excitotoxicity than those from WT mice and the EP(3) receptor antagonist did not attenuate the excitotoxicity. In transient focal ischaemia models, injection (i.p.) of an EP(3) antagonist reduced infarction, oedema and neurological dysfunction in WT mice, but not in mPGES-1 KO mice, which showed less injury than WT mice. EP(3) receptor agonist-induced augmentation of excitotoxicity in vitro was ameliorated by the Rho kinase inhibitor Y-27632 and Pertussis toxin. The Rho kinase inhibitor HA-1077 also ameliorated stroke injury in vivo. CONCLUSION AND IMPLICATIONS: Activity of mPGES-1 exacerbated stroke injury through EP(3) receptors and activation of Rho kinase and/or G(i). Thus, mPGES-1 and EP(3) receptors may be valuable therapeutic targets for treatment of human stroke.

Glutamate receptors: the cause or cure in perinatal white matter injury?

Glutamate toxicity from hypoxia-ischaemia during the perinatal period causes white matter injury that can result in long-term motor and intellectual disability. Blocking ionotropic glutamate receptors (GluRs) has been shown to inhibit oligodendrocyte injury in vitro, but GluR antagonists have not yet proven helpful in clinical studies. The opposite approach of activating GluRs on developing oligodendrocytes shows promise in experimental studies on rodents as reported by Jartzie et al., in this issue. Group I metabotropic glutamate receptors (mGluRs) are expressed transiently on developing oligodendrocytes in humans during the perinatal period, and the blood-brain-barrier permeable agonist of group I mGluRs, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD), reduces white matter damage significantly in a rat model of perinatal hypoxia-ischaemia. The results suggest drugs activating this class of GluRs could provide a new therapeutic approach for preventing cerebral palsy and other neurological consequences of diffuse white matter injury in premature infants.

Polychlorinated biphenyls PCB 153 and PCB 126 impair the glutamate-nitric oxide-cGMP pathway in cerebellar neurons in culture by different mechanisms.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. Perinatal exposure to PCB 153 or PCB 126 impairs the glutamate-nitric oxide-cGMP pathway in cerebellum in vivo and learning ability in adult rats. The aims of this work were: (1) to assess whether long-term exposure of primary cultures of cerebellar neurons to PCB 153 or PCB 126 reproduces the impairment in the function of the glutamate-nitric oxide-cGMP pathway found in rat cerebellum in vivo; (2) to provide some insight on the steps of the pathway affected by these PCBs; (3) to assess whether the mechanisms of interference of the pathway are different for PCB 126 and PCB 153. Both PCB 153 and PCB 126 increase basal levels of cGMP by different mechanisms. PCB 126 increases the amount of soluble guanylate cyclase while PCB 153 does not. PCB 153 reduces the amount of calmodulin while PCB 126 does not. Also both PCBs impair the function of the glutamate-nitric oxide-cGMP pathway by different mechanisms, PCB 153 impairs nitric oxide-induced activation of soluble guanylate cyclase and increase in cGMP while PCB 126 does not. PCB 126 reduces NMDA-induced increase in calcium while PCB 153 does not. When PCB 153 and PCB 126 exhibit the same effect, PCB 126 was more potent than PCB 153, as occurs in vivo.

Adipose stromal cells-conditional medium protected glutamate-induced CGNs neuronal death by BDNF.

The delivery of factors secreted by adipose stromal cells (ASC) to the brain may be a viable neuroprotective therapeutic option. In this study, we investigated the bioactivity of ASC-conditioned medium (ASC-CM) in glutamate-induced neurotoxicity and found that the ASC-CM significantly blocked glutamate neurotoxicity. We identified the brain-derived neurotrophic factor (BDNF) in the ASC-CM by using Western blot and demonstrated that this activity was critical for the neuroprotective effect of ASC-CM in excytotoxicity models. Furthermore, inactivating BDNF also attenuated the suppression by ASC-CM of glutamate-induced caspase-3 activity, but not p38 phosphorylation. These findings suggest that among ASC secrete a potent combination of factors, BDNF play a major role in neuroprotection against excytotoxicity.

NP031112, a thiadiazolidinone compound, prevents inflammation and neurodegeneration under excitotoxic conditions: potential therapeutic role in brain disorders.

Inflammation and neurodegeneration coexist in many acute damage and chronic CNS disorders (e.g., stroke, Alzheimer's disease, Parkinson's disease). A well characterized animal model of brain damage involves administration of kainic acid, which causes limbic seizure activity and subsequent neuronal death, especially in the CA1 and CA3 pyramidal cells and interneurons in the hilus of the hippocampus. Our previous work demonstrated a potent anti-inflammatory and neuroprotective effect of two thiadiazolidinones compounds, NP00111 (2,4-dibenzyl-[1,2,4]thiadiazolidine-3,5-dione) and NP01138 (2-ethyl-4-phenyl-[1,2,4]thiadiazolidine-3,5-dione), in primary cultures of cortical neurons, astrocytes, and microglia. Here, we show that injection of NP031112, a more potent thiadiazolidinone derivative, into the rat hippocampus dramatically reduces kainic acid-induced inflammation, as measured by edema formation using T2-weighted magnetic resonance imaging and glial activation and has a neuroprotective effect in the damaged areas of the hippocampus. Last, NP031112-induced neuroprotection, both in vitro and in vivo, was substantially attenuated by cotreatment with GW9662 (2-chloro-5-nitrobenzanilide), a known antagonist of the nuclear receptor peroxisome proliferator-activated receptor gamma, suggesting that the effects of NP031112 can be mediated through activation of this receptor. As such, these findings identify NP031112 as a potential therapeutic agent for the treatment of neurodegenerative disorders.

The role of group I metabotropic glutamate receptors in neuronal excitotoxicity in Alzheimer's disease.

Neurodegenerative diseases such as Huntington's disease, ischemia, and Alzheimer's disease (AD) are major causes of death. Recently, metabotropic glutamate receptors (mGluRs), a group of seven-transmembrane-domain proteins that couple to G-proteins, have become of interest for studies of pathogenesis. Group I mGluRs control the levels of second messengers such as inositol 1,4,5-triphosphate (IP3), Ca2+ ions and cAMP. They elicit the release of arachidonic acid via intracellular Ca2+ mobilization from intracellular stores such as mitochondria and endoplasmic reticulum. This facilitates the release of glutamate and could trigger the formation of neurofibrillary tangles, a pathological hallmark of AD. mGluRs regulate neuronal injury and survival, possibly through a series of downstream protein kinase and cysteine protease signaling pathways that affect mitochondrially mediated programmed cell death. They may also play a role in glutamate-induced neuronal death by facilitating Ca(II) mobilization. Hence, mGluRs have become a target for neuroprotective drug development. They represent a pharmacological path to a relatively subtle amelioration of neurotoxicity because they serve a modulatory rather than a direct role in excitatory glutamatergic transmission.

Improvements in an in-vitro assay for excitotoxicity by measurement of early gene (c-fos mRNA) levels.

Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15-30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.

Excitotoxicity: perspectives based on N-methyl-D-aspartate receptor subtypes.

Since excitotoxicity has been implicated in a variety of neuropathological conditions, understanding the pathways involved in this type of cell death is of critical importance to the future clinical treatment of many diseases. The N-methyl-D-aspartate (NMDA) receptor has become a primary focus of excitotoxic research because early studies demonstrated that antagonism of this receptor subtype was neuroprotective. However, initial pharmacological agents were not clinically useful due to the adverse effects of complete NMDA receptor blockade. Understanding the biochemical properties of the multitude of NMDA receptor subtypes offers the possibility of developing more effective and clinically useful drugs. With the discovery of the basis of heterogeneity of NMDA receptors through molecular biological approaches, many new potential therapeutic targets have been uncovered, and several model systems have been developed for the study of NMDA receptor-mediated cell death. This review discusses these models and the current understanding of the relationship between NMDA receptor subtypes and excitotoxicity.

Models of neural injury.

A variety of methods can be used experimentally to model neural injury, and thus study the process that represents the major pathophysiology underlying glaucomatous optic neuropathy. These methods vary in difficulty, relevance to human glaucoma, and time course. There is no perfect model, partly because our understanding of the glaucomas is still incomplete. Nonetheless, research into the mechanisms underlying the optic neuropathy of glaucoma (as well as possible new treatments) is advancing rapidly because of the availability of a wide variety of models of neural injury.