The objective of this project is to explore new strategies for the rational development of antiepileptic drugs based upon their interaction with neuronal ion channel systems. Cellular electrophysiological recording techniques are used to study drug modulation of neurotransmitter-gated and voltage-activated ion channels in brain slices, cultured neurons and heterologous cells transfected with cloned ion channel subunit genes. In the present reporting period, we continued our focus on glutamate receptors and glutamate receptor-mediated synaptic transmission. Functional activity of NMDA-type glutamate receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine. However, several other amino acids including D-serine can activate NMDA receptors when coapplied with glutamate. Although D-amino acids are prominent in bacteria, they generally are thought not to occur in mammals. However, high levelsof D-serine have recently been found in mammalian brain, suggesting that the amino acid could serve as an endogenous coagonist for NMDA receptors. We observed that D-amino acid oxidase, an enzyme that selectively degrades D-serine, greatly attenuates NMDA receptor-mediated neurotransmission as assessed using whole-cell patch-clamp recordings in cultured hippocampal neurons. The inhibitory effects of the enzyme were fully reversed by exogenously applied D-serine which did not by itself potentiate NMDA receptor-mediated synaptic responses. These observations indicate that D-serine is an endogenous modulator at the glycine site of NMDA receptors and that it may fully occupy this site at some functional synapses. Agents that modify the activity of D-serine metabolic enzymes, including the recently cloned mammalian serine racemase, could be useful to diminish excessive activation of NMDA receptors as may occur in epilepsy. Several widely used antiepileptic drugs are believed to act via use-dependent block of voltage-activated sodium channels. However, the way in which sodium channel modulation protects against seizures is not well understood. We examined the hypothesis that sodium channel blocking anticonvulsants, including phenytoin and zonisamide, act through inhibition of synaptic glutamate release. AMPA receptor-mediated spontaneous and evoked synaptic currents were recorded in CA1 pyramidal neurons of the rat hippocampal slice using whole-cell voltage clamp techniques. Clinically-relevant concentrations of zonisimide induced a large reduction in the frequency and amplitude of spontaneous excitatory synaptic currents without altering their kinetic properties. In contrast, the amplitude and frequency of miniature excitatory postsynaptic currents was unaffected by the drug, indicating that zonisimide acts presynaptically. Zonisimide had minimal effects on excitatory synaptic currents evoked by low frequency stimulation of the stratum radiatum. However, with high frequency stimulation there was a dramatic use-dependent inhibitory action on the release of excitatory transmitter. The widely used sodium channel blocking anticonvulsant phenytoin had similar actions. We conclude that sodium channel blocking anticonvulsants have a common action to reduce glutamate-mediated excitatory synaptic transmission through effects on glutamate release mechanisms.Studies were also continued examining glutamate receptor mediated neurotransmission and synaptic plasticity mechanisms in the amygdala, a key brain site for epileptogenesis in animal models and a common primary focus for seizures in human epilepsy. We previously demonstrated that a component of the excitatory synaptic response evoked in basolateral amygdala neurons by stimulation of the external capsule is mediated by kainate receptors containing the GluR5 subunit. Moreover, we observed that kainate receptor activation induces a novel form of NMDA receptor-independent enduring synaptic facilitation. We now demonstrate using in situ hybridization histochemistry that GluR5 kainate receptor mRNA is expressed at high levels in the basolateral amygdala; other kainate receptor subunit mRNAs were expressed at lower levels. GluR5 kainate receptors may be calcium permeable. To examine the hypothesis that calcium entry via GluR5 kainate receptors mediates GluR5 kainate receptor-dependent enduring synaptic facilitation, amygdala slices were treated with the membrane permeant calcium chelator BAPTA-AM. Slices exposed to BAPTA-AM failed to show GluR5 kainate receptor-mediated synaptic plasticity although synaptic responses were largely unaffected. Fura-2 imaging confirmed that BAPTA-AM produced a substantial reduction in intracellular calcium. We conclude that GluR5 containing kainate receptors mediate a novel form of calcium-dependent enduring synaptic facilitation. We propose that GluR5 kainate receptors could serve as mediators of epileptic hyperexcitability and epileptogenesis, such as occurs in response to kainate receptor agonists including the neurotoxins kainate and domoate, and we further suggest that drugs that block GluR5 kainate receptors could be useful in the prevention and treatment of some forms of epilepsy.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Intramural Research (Z01)
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Reddy, Doodipala S; Rogawski, Michael A (2010) Neurosteroids as endogenous regulators of seizure susceptibility and use in the treatment of epilepsy. Epilepsia 51:84
Gasior, Maciej; White, Natalie A; Rogawski, Michael A (2007) Prolonged attenuation of amygdala-kindled seizure measures in rats by convection-enhanced delivery of the N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA. J Pharmacol Exp Ther 323:458-68
Gasior, Maciej; French, Amy; Joy, Michelle T et al. (2007) The anticonvulsant activity of acetone, the major ketone body in the ketogenic diet, is not dependent on its metabolites acetol, 1,2-propanediol, methylglyoxal, or pyruvic acid. Epilepsia 48:793-800
Rogawski, Michael A (2006) Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy Res 69:273-94
Gasior, Maciej; Rogawski, Michael A; Hartman, Adam L (2006) Neuroprotective and disease-modifying effects of the ketogenic diet. Behav Pharmacol 17:431-9
Loscher, Wolfgang; Schmidt, Dieter (2006) New Horizons in the development of antiepileptic drugs: Innovative strategies. Epilepsy Res 69:183-272
Motamedi, Gholam K; Salazar, Patricia; Smith, Eric L et al. (2006) Termination of epileptiform activity by cooling in rat hippocampal slice epilepsy models. Epilepsy Res 70:200-10
Rogawski, Michael A (2006) Molecular targets versus models for new antiepileptic drug discovery. Epilepsy Res 68:22-8
Biagini, Giuseppe; Baldelli, Enrica; Longo, Daniela et al. (2006) Endogenous neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy. Exp Neurol 201:519-24
Winitsky, Steve O; Gopal, Thiru V; Hassanzadeh, Shahin et al. (2005) Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro. PLoS Biol 3:e87

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