Brian S. Meldrum

Calcium Overload in Selectively Vulnerable Neurons of the Hippocampus during and after Ischemia: An Electron Microscopy Study in the Rat

Light and electron microscopy has been used to study the cytopathological changes in the rat hippocampus directly after a 30-min period of forebrain ischemia and after 30 or 120 min of reperfusion. The fine structural localization of calcium has been demonstrated using the oxalate/pyroantimonate procedure. Cellular changes considered typical of ischemia (swelling of astrocytic processes, distention of mitochondria, condensation of cytoplasm, “ischemic cell change”) are most prominent after 30 min of reperfusion. At this time, dense calcium pyroantimonate deposits are evident in swollen mitochondria in pyramidal and hilar neurons. After 120 min of reperfusion, substantial restitution has occurred; most mitochondria appear normal and there are few calcium deposits. However, a small number of selectively vulnerable neurons (hilar and pyramidal neurons) show dense condensation (ischemic cell change) with multiple vacuoles containing calcium deposits. The role of excessive calcium entry and mitochondrial calcium overload during the reperfusion period in determining the death of selectively vulnerable neurons is discussed.

Molecular Targets for Antiepileptic Drug Development

SummaryThis review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, α2-δ voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABAA receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABAB and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated currentIh; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABAA receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies.

Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid

The anticonvulsant activity of 2-amino-7-phosphonoheptanoic acid (2APH) (an antagonist of excitation induced by N-methyl-D-aspartic acid) was studied against N-methyl-DL-aspartic acid (NMDLA), kainic acid, 3-mercaptopropionic acid (3MPA), thiosemicarbazide (TSC), quinolinic acid, bicuculline, picrotoxin and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in Swiss S mice. 2APH, 0.33 mM/kg i.p., antagonizes convulsions induced by NMDLA, 3MPA, TSC, DMCM and picrotoxin but not those produced by the other convulsants. It is proposed that an aspartergic component may contribute to the development of convulsions after 3MPA, TSC, DMCM and picrotoxin.

The non-NMDA antagonists, NBQX and GYKI 52466, protect against cortical and striatal cell loss following transient global ischaemia in the rat

The cerebroprotective action of non-NMDA receptor blockade has been assessed in a model of transient global ischaemia using NBQX, 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, and GYKI 52466, 1-(amino-phenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine. HCl. In Wistar rats, prior cauterisation of the vertebral arteries was followed by occlusion of the common carotid arteries for 20 min, with a 7 day survival period before histological evaluation. NBQX, 40 mg/kg, or GYKI 52466, 40 mg/kg, was administered intravenously starting directly after the end of carotid occlusion and ending 3 h later. Both compounds produced significant protection against selective cell loss in the striatum and cortex. Less consistent changes were seen in the hippocampus; protection by NBQX was significant in CA3 but neither compound produced significant protection in CA1. This pattern of protection is interpreted in terms of a blockade of glutamates action at non-NMDA receptors limited to the initial 3 h of reperfusion.

An adenosine analogue, 2-chloroadenosine, protects against long term development of ischaemic cell loss in the rat hippocampus

Delayed ischaemic damage was assessed by light microscopy following 10 min of incomplete forebrain ischaemia and 7 days of reperfusion. Iterative focal injections of 2-chloroadenosine, a stable analogue of adenosine, protect against selective hippocampal CA1 loss (P less than 0.01), when given either immediately before ischaemia and after 4 and 10 h of reperfusion or after 1 min, 4 h and 10 h of reperfusion. Delayed focal injection of 2-chloroadenosine after 10 and 24 h of reperfusion fails to protect against CA1 cell loss. The mechanism of cerebroprotection may involve attenuation of excitatory neurotransmission.

The anticonvulsant effect of the non-NMDA antagonists, NBQX and GYKI 52466, in mice

The excitatory amino acid antagonists, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline) and GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) that act on non-NMDA receptors, provide potent anticonvulsant protection against AMPA [RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-induced seizures in Swiss mice and against sound-induced seizures in seizure-susceptible DBA/2 mice. Maximal anticonvulsant protection is observed 5-30 min after the i.p. administration of NBQX and 5-15 min after the i.p. administration of GYKI 52466 in DBA/2 mice. The ED50 values for the protection against AMPA-induced seizures by NBQX (30 min, i.p.) and GYKI 52466 (15 min, i.p.) are 23.6 (11.6-48.0) and 18.5 (11.5-29.5) mumol/kg, respectively. The ED50 values at 15 min for the protection against sound-induced seizures in DBA/2 mice are 31.3 (24.9-39.4) mumol/kg (NBQX, i.p.), 37.8 (21.2-67.4) mumol/kg (NBQX, i.v.) and 13.7 (11.5-16.5) mumol/kg (GYKI 52466, i.p.). In DBA/2 mice the therapeutic index (ratio of ED50 values for impaired rotarod performance and anticonvulsant action) is 6.6 for NBQX (15 and 30 min, i.p.) and 2.0 for GYKI 52466 (15 min, i.p.).

Antiepileptic action of excitatory amino acid antagonists in the photosensitive baboon, papio papio

Antagonists of excitation induced by dicarboxylic amino acids have been evaluated for acute anticonvulsant activity in baboons, Papio papio, with photosensitive epilepsy. 2-Amino-7-phosphonoheptanoic acid, 1 mmol/kg, i.v., abolishes myoclonic responses for 5 h. Less prolonged protection is seen after cis-2,3-piperidine-dicarboxylic acid, 1-3.3 mmol/kg; 2-amino-5-phosphonovaleric acid, 1-3.3 mmol/kg; or glutamic acid diethyl ester, 1-3.3 mmol/kg. Toxic side-effects are prominent after the latter two compounds. Antagonists of excitation due to N-methyl-D-aspartate possess acute anticonvulsant activity in a wider range of models epilepsy.

Anticonvulsant action of ethanolamine-O-sulphate and di-n-propylacetate and the metabolism of γ-aminobutyric acid (GABA) in mice with audiogenic seizures

Abstract Mice susceptible to ‘audiogenic’ seizures (DBA/2, 21–25 days old) were treated with either di-n-propylacetate. DPA, (200–600 mg/kg, intraperitoneally) or ethanolamine-O-sulphate, EOS, (7.5–15 mg/kg, intracerebroventricularly). Motor behaviour was not moditied 45 min after DPA (except for slight changes after 600 mg/kg). Seizure responses to auditory stimulation were severely reduced after DPA 400 mg/kg, and totally absent after 600 mg/kg. Brain γ-aminobutyric acid (GABA) concentrations were unchanged after DPA 200–400 mg/kg, but increased by 57% after 600 mg/kg. The latter dose inhibited brain GABA-transaminase (4-aminobutyrate-2-oxoglutarate aminotransferase) activity by 33%. Kinetic studies with brain homogenates failed to show inhibition of GABA-transaminase activity by DPA (5–15 mM), but demonstrated inhibition of succinic Semialdehyde dehydrogenase by substrate competition. Mice tested 24 hr after EOS injection showed mild to moderate alaxia and were completely protected against ‘audiogenic’ seizures. Brain GABA concentration was increased 4–10 fold. GABA-transaminase activity was inhibited by 54–58% There was no inhibition of succinic Semialdehyde dehydrogenase activity.

Glutamate metabotropic receptors as targets for drug therapy in epilepsy

Metabotropic glutamate (mGlu) receptors have multiple actions on neuronal excitability through G-protein-linked modifications of enzymes and ion channels. They act presynaptically to modify glutamatergic and gamma-aminobutyric acid (GABA)-ergic transmission and can contribute to long-term changes in synaptic function. The recent identification of subtype-selective agonists and antagonists has permitted evaluation of mGlu receptors as potential targets in the treatment of epilepsy. Agonists acting on group I mGlu receptors (mGlu1 and mGlu5) are convulsant. Antagonists acting on mGlu1 or mGlu5 receptors are anticonvulsant against 3,5-dihydroxyphenylglycine (DHPG)-induced seizures and in mouse models of generalized motor seizures and absence seizures. The competitive, phenylglycine mGlu1/5 receptor antagonists generally require intracerebroventricular administration for potent anticonvulsant efficacy but noncompetitive antagonists, e.g., (3aS,6aS)-6a-naphthalen-2-ylmethyl-5-methyliden-hexahydrocyclopenta[c]furan-1-on (BAY36-7620), 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) block generalized seizures with systemic administration. Agonists acting on group II mGlu receptors (mGlu2, mGlu3) to reduce glutamate release are anticonvulsant, e.g., 2R,4R-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC], (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268). The classical agonists acting on group III mGlu receptors such as L-(+)-2-amino-4-phosphonobutyric acid, and L-serine-O-phosphate are acutely proconvulsant with some anticonvulsant activity. The more recently identified agonists (R,S)-4-phosphonophenylglycine [(R,S)-PPG] and (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG] and (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid [ACPT-1] are all anticonvulsant without proconvulsant effects. Studies in animal models of kindling reveal some efficacy of mGlu receptor ligands against fully kindled limbic seizures. In genetic mouse models, mGlu1/5 antagonists and mGlu2/3 agonists are effective against absence seizures. Thus, antagonists at group I mGlu receptors and agonists at groups II and III mGlu receptors are potential antiepileptic agents, but their clinical usefulness will depend on their acute and chronic side effects. Potential also exists for combining mGlu receptor ligands with other glutamatergic and non-glutamatergic agents to produce an enhanced anticonvulsant effect. This review also discusses what is known about mGlu receptor expression and function in rodent epilepsy models and human epileptic conditions.

Amino Acid Neurotransmitters and New Approaches to Anticonvulsant Drug Action

Summary: Amino acids provide the most universal and important inhibitory (γ‐aminobutyric acid (GABA), glycine) and excitatory (glutamate, aspartate, cysteic acid, cysteine sulphinic acid) neurotransmitters in the brain. An anticonvulsant action may be produced (1) by enhancing inhibitory (GABAergic) processes, and (2) by diminishing excitatory transmission. Possible pharmacological mechanisms for enhancing GABA‐mediated inhibition include (1) GABA agonist action, (2) GABA prodrugs, (3) drugs facilitating GABA release from terminals, (4) inhibition of GABA‐transaminase, (5) allosteric enhancement of the efficacy of GABA at the receptor complex, (6) direction action on the chloride ionophore, and (7) inhibition of GABA reuptake. Examples of these approaches include the use of irreversible GABA‐transaminase inhibitors, such as γ‐vinyl GABA, and the development of anticonvulsant β‐carbolines that interact with the “benzodiazepine receptor.” Pharmacological mechanisms for diminishing excitatory transmission include (1) enzyme inhibitors that decrease the maximal rate of synthesis of glutamate or asparate, (2) drugs that decrease the synaptic release of glutamate or aspartate, and (3) drugs that block the post‐synaptic action of excitatory amino acids. Compounds that selectively antagonise excitation due to dicarboxylic amino acids have recently been developed. Those that selectively block excitation produced by N‐methyl‐D‐aspartate (and aspartate) have proved to be potent anticonvulsants in many animal models of epilepsy. This provides a novel approach to the design of anticonvulsant drugs.