Glutamate Receptor Antagonist NBQX Research Articles

NMDA receptors in the insular cortex modulate cardiovascular and autonomic but not neuroendocrine responses to restraint stress in rats

The insular cortex (IC) is a brain structure involved in physiological and behavioural responses during stressful events. However, the local neurochemical mechanisms involved in control of stress responses by the IC are poorly understood. Thus, this study aimed to investigate the involvement of glutamatergic neurotransmission within the IC in cardiovascular, autonomic and neuroendocrine responses to an acute session of restraint stress. For this, the selective NMDA glutamate receptor antagonist LY235959 (1 nmol/100 nL) or the selective non-NMDA glutamate receptor antagonist NBQX (1 nmol/100 nL) were microinjected into the IC 10 min before the onset of the 60 min session of restraint stress. We observed that the antagonism of NMDA receptors within the IC enhanced the restraint-evoked increase in arterial pressure and heart rate, while blockade of non-NMDA receptors did not affect these cardiovascular responses. Spontaneous baroreflex analysis demonstrated that microinjection of LY235959 into the IC decreased baroreflex activity during restraint stress. The decrease in tail skin temperature during restraint stress was shifted to an increase in animals treated with the NMDA receptor antagonist. Nevertheless, the blockade of either NMDA or non-NMDA glutamate receptors within the IC did not affect the increase in circulating corticosterone levels during restraint stress. Overall, our findings provide evidence that IC glutamatergic neurotransmission, acting via local NMDA receptors, plays a prominent role in the control of autonomic and cardiovascular responses to restraint stress, but without affecting neuroendocrine adjustments.

Involvement of NMDA receptors in tremor expression in Aspa/Hcn1 double-knockout rats.

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspaem34Kyo/Hcn1A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspaem34Kyo/Hcn1em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.

Alcohol-Seeking Triggered by Discrete Pavlovian Cues is Invigorated by Alcohol Contexts and Mediated by Glutamate Signaling in the Basolateral Amygdala.

The environmental context in which a discrete Pavlovian conditioned stimulus (CS) is experienced can profoundly impact conditioned responding elicited by the CS. We hypothesized that alcohol-seeking behavior elicited by a discrete CS that predicted alcohol would be influenced by context and require glutamate signaling in the basolateral amygdala (BLA). Male, Long-Evans rats were allowed to drink 15% ethanol (v/v) until consumption stabilized. Next, rats received Pavlovian conditioning sessions in which a 10 s CS (15 trials/session) was paired with ethanol (0.2 ml/CS). Entries into a port where ethanol was delivered were measured. Pavlovian conditioning occurred in a specific context (alcohol context) and was alternated with sessions in a different context (non-alcohol context) where neither the CS nor ethanol was presented. At test, the CS was presented without ethanol in the alcohol context or the non-alcohol context, following a bilateral microinfusion (0.3 μl/hemisphere) of saline or the AMPA glutamate receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt) in the BLA (0, 0.3, or 1.0 μg/0.3 μl). The effect of NBQX (0, 0.3 μg/0.3 μl) in the caudate putamen (CPu) on CS responding in the non-alcohol context was also tested. The discrete alcohol CS triggered more alcohol-seeking behavior in the alcohol context than the non-alcohol context. NBQX in the BLA reduced CS responding in both contexts but had no effect in the CPu. These data indicate that AMPA glutamate receptors in the BLA are critical for alcohol-seeking elicited by a discrete CS and that behavior triggered by the CS is strongly invigorated by an alcohol context.

Involvement of non-NMDA glutamate receptors of the hypothalamic paraventricular nucleus in the cardiovascular response to the microinjection of noradrenaline into the dorsal periaqueductal gray area of rats

The dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. In a previous study, we showed that noradrenaline (NA) microinjected into the dPAG caused a vasopressin-mediated pressor response, involving a relay in the hypothalamic paraventricular nucleus (PVN). In the present study, we evaluated the involvement of ionotropic glutamate receptors within the PVN in the cardiovascular response to NA microinjection into the dPAG of unanesthetized rats. Microinjection of the selective NMDA glutamate receptor antagonist LY235959 (2nmol/100nL) unilaterally into the PVN did not affect the cardiovascular response evoked by microinjection of NA (15nmol/50nL) into the dPAG. On the other hand, unilateral PVN pretreatment with the non-NMDA glutamate receptor antagonist NBQX (2nmol/100nL) significantly reduced the pressor and cardiac response caused by microinjection of NA into the dPAG. In addition, bilateral PVN pretreatment with NBQX (2nmol/100nL) blocked the cardiovascular response to NA injected into the dPAG. In conclusion, the present results suggest that bilateral PVN activation of non-NMDA glutamate receptors mediates the vasopressin-related cardiovascular response to the microinjection of NA into the dPAG.

The glutamate receptor antagonist NBQX alleviates inflammation, pathology and gait abnormalities in rat antigen induced arthritis

The glutamate receptor antagonist NBQX alleviates inflammation, pathology and gait abnormalities in rat antigen induced arthritis

Paraventricular nucleus of the hypothalamus glutamate neurotransmission modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats

In the present study, the involvement of paraventricular nucleus of the hypothalamus (PVN) glutamate receptors in the modulation of autonomic (arterial blood pressure, heart rate and tail skin temperature) and neuroendocrine (plasma corticosterone) responses and behavioral consequences evoked by the acute restraint stress in rats was investigated. The bilateral microinjection of the selective non-NMDA glutamate receptor antagonist NBQX (2nmol/ 100nL) into the PVN reduced the arterial pressure increase as well as the fall in the tail cutaneous temperature induced by the restraint stress, without affecting the stress-induced tachycardiac response. On the other hand, the pretreatment of the PVN with the selective NMDA glutamate receptor antagonist LY235959 (2nmol/100nL) was able to increase the stress-evoked pressor and tachycardiac response, without affecting the fall in the cutaneous tail temperature. The treatment of the PVN with LY235959 also reduced the increase in plasma corticosterone levels during stress and inhibited the anxiogenic-like effect observed in the elevated plus-maze 24h after the restraint session. The present results show that NMDA and non-NMDA receptors in the PVN differently modulate responses associated to stress. The PVN glutamate neurotransmission, via non-NMDA receptors, has a facilitatory influence on stress-evoked autonomic responses. On the other hand, the present data point to an inhibitory role of PVN NMDA receptors on the cardiovascular responses to stress. Moreover, our findings also indicate an involvement of PVN NMDA glutamate receptors in the mediation of the plasma corticosterone response as well as in the delayed emotional consequences induced by the restraint stress.

N-Methyl- d-aspartate glutamate receptors in the hypothalamic paraventricular nucleus modulate cardiac component of the baroreflex in unanesthetized rats

In the present study, we investigated the role played by the hypothalamic paraventricular nucleus (PVN) in the modulation of cardiac baroreflex activity in unanesthetized rats. Bilateral microinjections of the nonselective neurotransmission blocker CoCl 2 into the PVN decreased the reflex bradycardic response evoked by blood pressure increases, but had no effect on reflex tachycardia evoked by blood pressure decreases. Bilateral microinjections of the selective NMDA glutamate receptor antagonist LY235959 into the PVN caused effects that were similar to those observed after microinjections of CoCl 2, decreasing reflex bradycardia without affecting tachycardic response. The microinjection of the selective non-NMDA glutamate receptor antagonist NBQX into the PVN did not affect the baroreflex activity. Also, the microinjection of L-glutamate into the PVN increased the reflex bradycardia, an effect opposed to that observed after PVN treatment with CoCl 2 or LY235959, and this effect of L-glutamate was blocked by PVN pretreatment with LY235959. LY235959 injected into the PVN after i.v. treatment with the selective β 1-adrenoceptor antagonist atenolol still decreased the reflex bradycardia. Taken together, our results suggest a facilitatory influence of the PVN on the bradycardic response of the baroreflex through activation of local NMDA glutamate receptors and a modulation of the cardiac parasympathetic activity.

BMAA selectively injures motor neurons via AMPA/kainate receptor activation

The toxin beta-methylamino- l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis–Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations (∼ 30 μM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca 2+] i rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

NMDA Receptors Mediate Neuron-to-Glia Signaling in Mouse Cortical Astrocytes

Chemical transmission between neurons and glial cells is an important element of integration in the CNS. Here, we describe currents activated by NMDA in cortical astrocytes, identified in transgenic mice that express enhanced green fluorescent protein under control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp either in slices or after gentle nonenzymatic mechanical dissociation. Acutely isolated astrocytes showed a three-component response to glutamate. The initial rapid component was blocked by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), which is an antagonist of AMPA receptors (IC50, 2 microM), and the NMDA receptor antagonist D-AP-5 blocked the later sustained component (IC50, 0.6 microM). The third component of glutamate application response was sensitive to D,L-threo-beta-benzyloxyaspartate, a glutamate transporter blocker. Fast application of NMDA evoked concentration-dependent inward currents (EC50, 0.3 microM); these showed use-dependent block by (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801). These NMDA-evoked currents were linearly dependent on membrane potential and were not affected by extracellular magnesium at concentrations up to 10 mM. Electrical stimulation of axons in layer IV-VI induced a complex inward current in astrocytes situated in the cortical layer II, part of which was sensitive to MK-801 at holding potential -80 mV and was not affected by the AMPA glutamate receptor antagonist NBQX. The fast miniature spontaneous currents were observed in cortical astrocytes in slices as well. These currents exhibited both AMPA and NMDA receptor-mediated components. We conclude that cortical astrocytes express functional NMDA receptors that are devoid of Mg2+ block, and these receptors are involved in neuronal-glial signal transmission.

Mechanisms contributing to the exacerbated epileptiform activity in hippocampal slices expressing a C-terminal truncated GABA B2 receptor subunit

GABAergic synaptic transmission plays an important role in the patterning of epileptiform activity. We have previously shown that global loss of GABA B receptor function due to transgenic deletion of the GABA B1 receptor subunit exacerbates epileptiform activity induced by pharmacological manipulations in hippocampal slices. Here we show that a similar hyperexcitable phenotype is observed in hippocampal slices prepared from a transgenic mouse expressing a GABA B2 receptor subunit lacking its C terminal tail (the ΔGB2-Ct mouse); a molecular manipulation that also produces complete loss of GABA B receptor function. Thus, epileptiform bursts that are sensitive to NMDA receptor antagonists (induced by either the GABA A receptor antagonist bicuculline (10 μM) or removal of extracellular Mg 2+) were significantly longer in duration in ΔGB2-Ct slices relative to WT slices. We now extend these observations to demonstrate that a stimulus train induced bursting (STIB) protocol also evokes significantly longer bicuculline sensitive bursts of activity in ΔGB2-Ct slices compared to WT. Furthermore, synchronous GABA A receptor-mediated potentials recorded in the presence of the potassium channel blocker 4-aminopyridine (4-AP, 100 μM) and the ionotropic glutamate receptor antagonists NBQX (20 μM) and D-AP5 (50 μM) were significantly prolonged in duration in ΔGB2-Ct versus WT slices. These data suggest that the loss of GABA B receptor function in ΔGB2-Ct hippocampal slices promotes depolarising GABA A receptor-mediated events, which in turn, leads to the generation of ictal-like events, which may contribute to the epilepsy phenotype observed in vivo.

Synaptic and membrane properties of neurons in the dorsomedial hypothalamus

Studies in intact rats have shown that the dorsomedial hypothalamus (DMH) plays a key role in generating stress-induced physiologic changes, including activation of the hypothalamic–pituitary–adrenal axis through direct projections to paraventricular hypothalamic nucleus (PVN). However, little is known about the cellular properties of DMH neurons. We employed whole-cell patch-clamp recording techniques to characterize membrane properties and spontaneous post-synaptic currents (PSCs) in DMH neurons, including those projecting to PVN (identified by prior injection of DiI into PVN), in rat hypothalamic slices. DMH neurons ( n=86 total) had uniform membrane properties. However, PVN-projecting neurons ( n=32) had higher action potential (AP) thresholds, and fired fewer APs in response to current injection. Spontaneous PVN-projecting neurons ( n=20) also fired APs at lower rates (4.8±0.6 Hz) than spontaneous neurons of unknown projection ( n=38; 7.3±1.1 Hz). Spontaneous PSCs were observed in all neurons: One population expressed rapid decay characteristics (1.5–2.0 ms) and was blocked by non-NMDA ionotropic glutamate receptor antagonists NBQX or CNQX. Remaining PSCs reversed near E Cl, were blocked by the GABA A receptor antagonists picrotoxin or bicuculline methiodide (BMI), and had longer decay time constants (4.5–6.0 ms) that were modulated by pentobarbital. Tetrodotoxin markedly reduced the frequency of PSCs sensitive to NBQX but not to BMI. Thus, DMH is made up of electrophysiologically similar neurons and PVN-projecting neurons are less excitable than neurons of unknown projection. Furthermore, as suggested by studies in intact rats, neurons in the DMH, including those projecting to the PVN, are regulated by tonic GABA A and non-NMDA glutamate receptor-mediated synaptic transmission.

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA) during physiological and ischemia-like conditions

Maintenance of low extracellular glutamate ([Glu]O) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by dl-threo-beta-benzyloxyaspartate (dl-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of dl-TBOA for 48h was found to induce significant cell death in all hippocampal regions, with EC50 values ranging from 38 to 48μM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of dl-TBOA (100μM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10μM) dose of dl-TBOA during OGD, but not during the subsequent 48h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death.

Primary insults to the brain can initiate glutamate release that may result in excitotoxicity followed by neuronal cell death. This secondary process is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in vivo and requires new gene expression. Neuronal cyclooxygenase-2 (COX2) expression is upregulated following brain insults, via glutamatergic and inflammatory mechanisms. The products of COX2 are bioactive prostanoids and reactive oxygen species that may play a role in neuronal survival. This study explores the role of neuronal COX2 in glutamate excitotoxicity using cultured cerebellar granule neurons (day 8 in vitro). Treatment with excitotoxic concentrations of glutamate or kainate transiently induced COX2 mRNA (two- and threefold at 6 h, respectively, p < 0.05, Dunnett) and prostaglandin production (five- and sixfold at 30 min, respectively, p < 0.05, Dunnett). COX2 induction peaked at toxic concentrations of these excitatory amino acids. Surprisingly, NMDA, L-quisqualate, and trans-ACPD did not induce COX2 mRNA at any concentration tested. The glutamate receptor antagonist NBQX (5 microM, AMPA/kainate receptor) completely inhibited kainate-induced COX2 mRNA and partially inhibited glutamate-induced COX2 (p < 0.05, Dunnett). Other glutamate receptor antagonists, such as MK-801 (1 microM, NMDA receptor) or MCPG (500 microM, class 1 metabotropic receptors), partially attenuated glutamate-induced COX2 mRNA. These antagonists all reduced steady-state COX2 mRNA (p < 0.05, Dunnett). To determine whether COX2 might be an effector of excitotoxic cell death, cerebellar granule cells were pretreated (24 h) with the COX2-specific enzyme inhibitor, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2((5)H)-furanone) prior to glutamate challenge. DFU (1 to 1000 nM) completely protected cultured neurons from glutamate-mediated neurotoxicity. Approximately 50% protection from NMDA-mediated neurotoxicity, and no protection from kainate-mediated neurotoxicity was observed. Therefore, glutamate-mediated COX2 induction contributes to excitotoxic neuronal death. These results suggest that glutamate, NMDA, and kainate neurotoxicity involve distinct excitotoxic pathways, and that the glutamate and NMDA pathways may intersect at the level of COX2.

Elevation of intradendritic sodium concentration mediated by synaptic activation of metabotropic glutamate receptors in cerebellar Purkinje cells.

Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.

Role of glutamate receptors in transmission of vagal cardiac input to neurones in the nucleus tractus solitarii in dogs.

1. Vagal afferent input from cardiac mechanoreceptors excites neurones in the nucleus tractus solitarii (NTS), but discharge patterns evoked by physiological activation of pressure-sensitive cardiac mechanoreceptors have not been studied in vivo. The role of glutamate receptor subtypes in transmission of afferent activity to the NTS neurones has not been determined. The present study therefore has two aims: first, to characterise the discharge patterns of neurones in the NTS that receive pressure-sensitive vagal cardiac receptor input and second, to determine the roles of ionotropic glutamate receptor subtypes in the transmission of this putative cardiac mechanoreceptor-related activity to NTS neurones. 2. Pulse-synchronous activity of neurones in the NTS evoked by vagal afferent input was recorded extracellularly in an anaesthetised dog model using multibarrel glass electrodes, which allowed picoejection of the glutamate receptor antagonists NBQX or AP5 to block either non-NMDA or NMDA receptors, respectively, during the neuronal recording. Pressure sensitivity of the recorded neurones was examined by monitoring their response to a small increase in arterial blood pressure. Selective pressure activation of carotid sinus baroreceptors in an isolated sinus or selective denervation of aortic baroreceptors were used to test for convergent excitation of the neurones by arterial baroreceptors. 3. Pulse-synchronous cardiac-related neuronal activity recorded from neurones in both the right and left NTS was eliminated following section of the left (n = 17) or right (n = 1) vagus nerves. No spontaneous, non-pulsatile activity was observed in these neurones before or after vagotomy. Activity transmitted via left vagal afferents was found to be sensitive to changes in arterial blood pressure. In these neurones, activity was blocked in 13 of 17 neurones by picoejection of NBQX, with the remainder requiring both NBQX and AP5. None of the cardiac-related neurones responded to activation of carotid baroreceptors or denervation of aortic baroreceptors, indicating no convergence of activity from carotid baroreceptors or aortic baroreceptors with pressure thresholds of approximately 130 mmHg or less. 4. The results suggest that vagal pressure-sensitive afferent input from cardiac mechanoreceptors is transmitted primarily by left vagal afferent fibres via non-NMDA receptors to neurones in both the ipsilateral and contralateral NTS. NMDA receptors were also found to have a role in the activation of a small subpopulation of neurones.

P.6.097 On the quality of diagnostics of border neuropsychic disorders in working women

Maintenance of low extracellular glutamate ([Glu]O) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by dl-threo-beta-benzyloxyaspartate (dl-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of dl-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC50 values ranging from 38 to 48 μM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of dl-TBOA (100 μM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 μM) dose of dl-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

Evaluation of Anticonvulsant Drugs for Soman-Induced Seizure Activity

This report describes three lines of organophosphorus (OP) nerve-agent anticonvulsant studies: (a) a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, (b) a drug-screening effort to determine whether clinically useful antiepileptic drugs can modulate soman-induced seizures in rats, and (c) an advanced testing effort in which anticholinergic compounds are evaluated in comparison to the current anticonvulsant treatment (i.e., diazepam) in guinea pigs. Electroencephalographic (EEG) recordings were used in all studies. Basic studies were conducted in rats pretreated with HI-6 and challenged with 1.6 x the median lethal dose (LD 50 ) soman. Antimuscarinic compounds were extremely effective in blocking (pretreatment) or terminating soman seizures when given 5 min after seizure onset; however, significantly higher doses were required when treatment was delayed >10 min, and some antimuscarinic compounds lost anticonvulsant efficacy when treatment was delayed 40 min. Diazepam blocked seizure onset, yet seizures could recur after an initial period of anticonvulsant effect at doses ≤2.5 mg/kg. Diazepam could terminate ongoing seizures when given 5 min after seizure onset, but doses up to 20 mg/kg were ineffective when treatment was delayed for 40 min. The γ-aminobutyric acid (GABA) uptake inhibitor tiagabine was ineffective in blocking or terminating soman motor convulsions or seizures. The glutamate receptor antagonists NBQX, GYKI 52466, and memantine had weak or minimal antiseizure activity, even at doses that virtually eliminated signs of motor convulsions. The antinicotinic mecamylamine was ineffective in blocking or stopping seizure activity. Pretreatment with a narrow range of doses of the alpha-2-adrenergic agonist clonidine produced variable protection (40-60%) against seizure onset; treatment after seizure onset with clonidine was not effective. Screening studies in rats, using HI-6 pretreatment, showed that the clinically effective antiepileptic drugs pentobarbital and valproic acid were modestly effective in terminating seizures when given shortly (5 min) after seizure onset. In contrast, phenytoin, carbamazepine, felbamate, magnesium sulfate, ketamine, primidone, and lamotrigine were ineffective. Advanced studies of eight anticholinergic drugs compared with diazepam anticonvulsant therapy were performed in guinea pigs pretreated with pyridostigmine, challenged with soman (2 x LS 50 ), and immediately treated with 2-PAM Cl and atropine. The rank of potency for diazepam and eight antimuscarinic compounds, when administered 5 min after seizure onzet (scopolamine, , biperiden, benactyzine, trihexyphenidyl, azaprophen, procyclidine, aprophen, diazempam, atropine), closely resembles that obtained from the rat model.