Germán Sierra-Paredes

Extrasynaptic GABA and Glutamate Receptors in Epilepsy

Epilepsy is a neurological disorder in which normal brain function is disrupted as a consequence of intensive and synchronous burst activity from neuron assemblies. Epilepsies result from long-lasting plastic changes in the brain affecting neurotransmitter release, the properties of receptors and channels, synaptic reorganization and astrocyte activity. There is considerable evidence for alterations in glutamatergic and GABAergic synaptic transmission in the origin of the paroxysmal depolarization shifts that initiate epileptic activity. However, recent studies on non-synaptic transmission, receptor mobility and glia-neuron signaling pathways suggest that extrasynaptic GABA and glutamate receptors may play an important role in seizure initiation, maintenance and arrest. Extracellular aminoacids such as glutamate, aspartate, glycine and GABA seem to communicate neurons and glial cells acting primarily on extrasynaptic receptors. Synaptic and extrasynaptic glutamate and GABA receptors have been show to play different roles in neuronal excitability. NMDA and GABAA receptors expressed in a single neuron can be differentially regulated based on subcellular localization, and it has been proposed that distinct regulation of synaptic versus extrasynaptic receptors provides a mechanism for receptor adaptation in response to a variety of stimuli. Furthermore, glutamate and GABA receptors are highly mobile, and the number and composition of extrasynaptic receptors can be modulated by several factors. This review addresses recent advances in our understanding of the role of extrasynaptic receptors in epilepsy, suggesting that extrasynaptic receptors and their mechanisms of regulation are expected to be important pharmacological targets.

Ascomycin and FK506: Pharmacology and Therapeutic Potential as Anticonvulsants and Neuroprotectants

Ascomycin and FK506 are powerful calcium‐dependent serine/threonine protein phosphatase (calcineurin [CaN], protein phosphatase 2B) inhibitors. Their mechanism of action involves the formation of a molecular complex with the intracellular FK506‐binding protein‐12 (FKBP12), thereby acquiring the ability to interact with CaN and to interfere with the dephosphorylation of various substrates. Pharmacological studies of ascomycin, FK506, and derivatives have mainly been focused on their action as immunosuppressants and therapeutic use in inflammatory skin diseases, both in animal studies and in humans. CaN inhibitors have been also proposed for the treatment of inflammatory and degenerative brain diseases. Preclinical studies suggest, however, that ascomycin and its derivatives exhibit additional pharmacological activities. Ascomycin has been shown to have anticonvulsant activity when perfused into the rat hippocampus via microdialysis probes, and ascomycin derivatives may be useful in preventing ischemic brain damage and neuronal death. Their pharmacological action in the brain may involve CaN‐mediated regulation of gamma aminobutyric acid (GABA) and glutamate receptor channels, neuronal cytoskeleton and dendritic spine morphology, as well as of the inflammatory responses in glial cells. FK506 and ascomycin inhibit signaling pathways in astrocytes and change the pattern of cytokine and neurotrophin gene expression. However, brain‐specific mechanisms of action other than CaN inhibition cannot be excluded. CaN is a likely potential target molecule in the treatment of central nervous system (CNS) diseases, so that the therapeutic potential of ascomycin, FK506, and nonimmunosuppressant ascomycin derivatives as CNS drugs should be further explored.

Effect of ionotropic glutamate receptors antagonists on the modifications in extracellular glutamate and aspartate levels during picrotoxin seizures: a microdialysis study in freely moving rats

Our previous studies have shown a local decrease in glutamate and aspartate levels during seizures, induced by picrotoxin microdialysis in the hippocampus of chronic freely moving rats. In this paper, we study the effect of continuous hippocampal microperfusion of the NMDA, AMPA and kainate glutamate receptor inhibitors 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801); 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). We also examine the action of L(-)-threo-3-hydroxyaspartic acid (THA), a glutamate and aspartate reuptake blocker, on the modification of extracellular glutamate and aspartate levels induced by picrotoxin, using the microdialysis method in freely moving rats. We found that changes in extracellular hippocampal concentrations in both amino acids are prevented by NMDA, AMPA and kainate receptor inhibitors. Seizures elicited under DNQX also induce a transient increase in aspartate extracellular levels coincident with seizure time. L(-)-threo-3-hydroxyaspartic acid increased the basal extracellular concentrations of both amino acids, but did not prevent the seizure-related decrease. Our results suggest that glutamate, the major neurotransmitter at the synaptic level, may also play an important role in non-synaptic transmission during seizures.

Microperfusion of picrotoxin in the hippocampus of chronic freely moving rats through microdialysis probes: a new method of induce partial and secondary generalized seizures

In this study we investigated the threshold and latency of seizures in a new model of chemically induced partial seizures, with or without secondary generalization. We performed repeated experiments with a maximum of 20 between day seizures on each individual rat over a period up to 6 months, with resting intervals ranging from 3 to 15 days depending on the severity of seizures. Seizures were induced by microperfusion of variable doses of picrotoxin starting at 100 microM in each animal and increasing in steps of 25 microM through microdialysis probes introduced in the hippocampus of freely moving rats. The seizures were registered in a Holter-EEG and videotaped. Only one picrotoxin dose was perfused during each experimental session. Threshold was defined as the minimum dose of picrotoxin which induced a type of seizure after a 5 min period of perfusion. Latency was defined as the period from the end of the microperfusion period to the onset of electro-behavioral seizures. Patterns of seizures induced in different experimental sessions showed a dose-dependent variation ranging from arrest behavior (absence-like) to rapidly recurring seizures. A permanent seizure threshold for each type of seizure was observed in repeated experiments within individual rats, but when compared with different animals, significant variations in threshold were observed. A long latency period (14-41 min) was observed, unrelated to threshold or seizure type. Since the method can induce separate seizures with constant seizure threshold in experiment rats over a testing period as long as 6 months, the described method offers a new way to test a large number of antiepileptic drugs on the same animals.

Seizures induced by in vivo latrunculin a and jasplakinolide microperfusion in the rat hippocampus.

The molecular basis for developing epilepsy remains under debate. It is hypothesized that increased excitatory synaptic activity might activate the N-methyl-D-aspartate receptor/Ca2+ transduction pathway, which induces long-lasting plasticity changes leading to recurrent epileptiform discharges. To determine if these effects are caused by disruption of F-actin in the dendritic spines, we have perfused the hippocampus of conscious rats with the F-actin-depolymerizing agent latrunculin A and the actin filament stabilizer jas plakinolide. Single perfusions of latrunculin A and jasplakinolide decrease and increase picrotoxin seizure threshold, respectively. Repeated perfusions of both latrunculin A and jasplakinolide induce epileptic seizures and a long-term increase in neuronal excitability. These results suggest that actin disruption might not be just a consequence but also a possible cause of epileptic seizures. We propose a new experimental model in rats to study the biochemical changes that might lead to chronic seizures and a method for testing new antiepileptic drugs.

Effect of eslicarbazepine acetate (BIA 2-093) on latrunculin A-induced seizures and extracellular amino acid concentrations in the rat hippocampus

PURPOSE Eslicarbazepine acetate (ESL, BIA 2-093) is a novel antiepileptic drug endowed with an anticonvulsant potency similar to that of carbamazepine, and shares with carbamazepine and oxcarbazepine the capability to inhibit voltage-gated sodium channels. ESL is efficacious against maximal electroshock seizure-induced seizures, protects against picrotoxin-induced seizures in mice and rats, and prevents development of kindling in rats. In vivo, latrunculin A microperfusion in the rat hippocampus induces acute epileptic seizures and long-term biochemical changes leading to decreased picrotoxin seizure threshold and spontaneous seizures. We have tested the effect of ESL on latrunculin A-induced seizures, and its effect on the changes in extracellular amino acid levels induced by latrunculin A. METHODS Rat hippocampus was continuously perfused with a latrunculin A solution (4 microM) through CMA/12 microdialysis probes at a flow rate of 2 microl/min during 8 h with continuous EEG and videotape recording for 3 consecutive days. The same protocol was repeated after oral administration of ESL (3, 10 and 30 mg/kg). Samples from the microdialysate were collected and analyzed by HPLC using pre-column derivatization with 6 aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescence detection. RESULTS After the administration of 3 mg/kg of ESL, seizures were completely suppressed in the 66.7% of the rats. 10 and 30 mg/kg of ESL did completely suppressed seizures in the 100% of the animals studied. Hippocampal extracellular levels of glutamate, glycine and aspartate were significantly increased during latrunculin A microperfusion, while GABA levels remained unchanged. At the doses studied, ESL reversed the increases in extracellular glutamate and aspartate concentrations to basal levels and significantly reduced glycine levels. CONCLUSIONS ESL, at oral doses of 3, 10 and 30 mg/kg, shows an excellent anticonvulsant effect against seizures induced by latrunculin A microperfusion in the rat, and prevents the increases in glutamate and aspartate induced by latrunculin A.

Extracellular amino acids in the rat hippocampus during picrotoxin threshold seizures in chronic microdialysis experiments

The relation between changes in the concentrations of some of the neuroactive extracellular amino acids (glutamate, aspartate, gamma-aminobutyric acid, glycine and taurine) and epileptic seizures has been tested in a new experimental model of seizures induced by picrotoxin microdialysis in chronic freely moving rats. During ictal discharges (paroxysmal electroencephalographic discharges associated with behavioral seizures), a significant decrease in the levels of extracellular aspartate and glutamate was observed. However, no changes were found during the interictal discharges (paroxysmal electroencephalographic discharges, without concomitant seizures). Our results suggest that modifications in extracellular aspartate and glutamate may be related to neuronal synchronization rather than to paroxysmal activity, supporting the neurophysiological differences between non-ictal and ictal paroxysmal activity.

Anticonvulsant effect of eslicarbazepine acetate (BIA 2-093) on seizures induced by microperfusion of picrotoxin in the hippocampus of freely moving rats.

Eslicarbazepine acetate (BIA 2-093, S-(-)-10-acetoxy-10,11-dihydro-5H-dibenzo/b,f/azepine-5-carboxamide) is a novel antiepileptic drug, now in Phase III clinical trials, designed with the aim of improving efficacy and safety in comparison with the structurally related drugs carbamazepine (CBZ) and oxcarbazepine (OXC). We have studied the effects of oral treatment with eslicarbazepine acetate on a whole-animal model in which partial seizures can be elicited repeatedly on different days without changes in threshold or seizure patterns. In the animals treated with threshold doses of picrotoxin, the average number of seizures was 2.3+/-1.2, and average seizure duration was 39.5+/-8.4s. Pre-treatment with a dose of 30 mg/kg 2h before picrotoxin microperfusion prevented seizures in the 75% of the rats. Lower doses (3 and 10mg/kg) did not suppress seizures, however, after administration of 10mg/kg, significant reductions in seizures duration (24.3+/-6.8s) and seizure number (1.6+/-0.34) were found. No adverse effects of eslicarbazepine acetate were observed in the behavioral/EEG patterns studied, including sleep/wakefulness cycle, at the doses studied.

Effect of extracellular long-time microperfusion of high concentrations of glutamate and glycine on picrotoxin seizure thresholds in the hippocampus of freely moving rats.

The effect of high concentrations of glutamate and glycine on picrotoxin seizure thresholds was investigated by perfusion through microdialysis probes in the hippocampus of freely moving rats. Microperfusion of glutamate at concentrations up to 1 mM, produced no changes in behavior or basal EEG recordings, but microperfusion of 200 microM glutamate was sufficient to lower the picrotoxin seizure threshold down to 50% in 60% of the animals studied and produced an increase of 180+/-23% in seizure duration. Microperfusion of 1 mM glutamate reduced seizure threshold in all animals, and markedly prolonged seizure duration (230+/-30%). Microperfusion of 200 microM or 1 mM glycine lowered picrotoxin seizure thresholds down to 50% in 70% of the animals and lengthened seizure duration up to 176+/-43%. Continuous microperfusion of the antagonist for the glycine binding site in NMDA receptors 5,7-dichlorokynurenic acid (100 microM) reversed the effect of both glutamate (1 mM) and glycine (1 mM) and suppressed seizures completely in 90% of the animals. These results indicate that although neurotoxicity is not achieved by perfusing glutamate and glycine at concentrations as high as 1 mM, neuronal excitability is modified by altering extracellular glutamate and glycine concentrations, and they suggest that glutamate-induced neuronal hyperexcitability is induced through mechanisms different from excitotoxicity.

Experimental spike-and-wave discharges induced by pentylenetetrazol and tolerance to repeated injections: an electrophysiological and biochemical study

This study was designed to obtain experimental data to correlate duration of spike-and-wave (SW) paroxysms with levels of pentylenetetrazol (PTZ) in several brain regions after intraperitoneal (i.p.) administration of subconvulsive doses of PTZ in Wistar rats. The influence of subconvulsive doses of PTZ on blood-brain barrier (BBB) permeability and tolerance of PTZ to repeated injections were also studied. Intraperitoneal administration of subconvulsive doses of PTZ (25 mg/kg) in single doses resulted in SW activity which accounted for 20% of the continuous electrical brain activity recorded during the first hour after i.p. administration. Brain PTZ levels (cortex, midbrain, cerebellum) were within the range 19.2-34.9 micrograms/g. Repeated doses of PTZ showed a significant decrease in SW activity with no change in PTZ brain levels. As PTZ bioavailability was the same after either a single dose or after repeated doses, the decrease in SW activity may be due to PTZ tolerance. No alterations in the BBB were induced by PTZ subconvulsive doses. The experimental data reported in this study may be useful to quantify modifications of biochemical parameters or to evaluate antiepileptic drugs.