Thalidomide protects against acute pentylenetetrazol and pilocarpine-induced seizures in mice.

Thalidomide was originally developed to treat primary neurological and psychiatric diseases. There are reports of anticonvulsant effects of thalidomide in rats and antiepileptic effects in patients. Hence, thalidomide (100, 200 and 400 mg/kg) was herein administered to mice to evaluate possible protection against seizures induced by the systemic administration of neurotoxins: 10 mg/kg of 4-aminopyridine (4-AP), 90 mg/kg of pentylenetetrazol (PTZ), or 380 mg/kg of pilocarpine. The effect of an NO and COX inhibitor (7-NI and ibuprofen, respectively) was also examined. The results show that thalidomide (1) induces the typical sedative effects, (2) has no anticonvulsant effect in mice treated with 4-AP, and (3) has anticonvulsant effect (400 mg/kg) in mice treated with PTZ and pilocarpine. It was found that 7-NI has an anticonvulsant effect in the pilocarpine model and that thalidomide's effect is not enhanced by its presence. However, thalidomide (200 mg/kg) plus 7-NI or ibuprofen tend to have a toxic effect in PTZ model. On the other hand, the combination of thalidomide and 7-NI or ibuprofen protects against pilocarpine-induced seizures. In conclusion, thalidomide did not exert an anticonvulsant effect for clonic-tonic type convulsions (4-AP), but it did so for seizures induced by PTZ and pilocarpine (representing absence seizures and status epilepticus, respectively). NO and prostaglandins were involved in the convulsive process elicited by pilocarpine.

Analysis of connexin expression during seizures induced by 4-aminopyridine in the rat hippocampus.

BACKGROUND: In epilepsy, seizures are generated by abnormal synchronous activity in neurons. In the rat hippocampus (HIP), epileptiform activity has been found to be associated with gap junctions (GJs). GJs are formed by the combination of two hemichannels, each composed of six connexins. At low doses, the convulsive drug 4-aminopyridine (4-AP) produces epileptiform activity without affecting glutamate levels; therefore, GJs could participate in its effect. Based on this argument, in this study, the expression of Cx 32, Cx 36 and Cx 43 protein and mRNA in the HIP of rats treated with 4-AP was evaluated. The evaluation of connexins was carried out by chemifluorescent immunoassay, semiquantitative RT-PCR and immunofluorescence to detect the amount and distribution of connexins and of cellular markers in the HIP and dentate gyrus (DG) of animals treated with NaCl and 4-AP in the right entorhinal cortex. In these animals, convulsive behavior and EEG signals were analyzed. RESULTS: The animals treated with 4-AP showed convulsive behavior and epileptiform activity 60 min after the administration. A significant increase in the protein expression of Cx 32, Cx 36 and Cx 43 was found in the HIP contralateral and ipsilateral to the site of 4-AP administration. A trend toward an increase in the mRNA of Cx 32 and Cx 43 was also found. An increase in the cellular density of Cx 32 and Cx 43 was found in the right HIP and DG, and an increase in the cellular density of oligodendrocytes in the DG and a decrease in the number of cells marked with NeuN were observed in the left HIP. CONCLUSIONS: Cx 32 and Cx 43 associated with oligodendrocytes and astrocytes had an important role in the first stages of seizures induced by 4-AP, whereas Cx36 localized to neurons could be associated with later stages. Additionally, these results contribute to our understanding of the role of connexins in acute seizures and allow us to direct our efforts to other new anticonvulsant strategies for seizure treatment.

The selective A-type K+ current blocker Tx3-1 isolated from the Phoneutria nigriventer venom enhances memory of naïve and Aß(25-35)-treated mice.

Potassium channels regulate many neuronal functions, including neuronal excitability and synaptic plasticity, contributing, by these means, to mnemonic processes. In particular, A-type K(+) currents (IA) play a key role in hippocampal synaptic plasticity. Therefore, we evaluated the effect of the peptidic toxin Tx3-1, a selective blocker of IA currents, extracted from the venom of the spider Phoneutria nigriventer, on memory of mice. Administration of Tx3-1 (i.c.v., 300 pmol/site) enhanced both short- and long-term memory consolidation of mice tested in the novel object recognition task. In comparison, 4-aminopyridine (4-AP; i.c.v., 30-300 pmol/site), a non-selective K(+) channel blocker did not alter long-term memory and caused toxic side effects such as circling, freezing and tonic-clonic seizures. Moreover, Tx3-1 (i.c.v., 10-100 pmol/site) restored memory of Aß25-35-injected mice, and exhibited a higher potency to improve memory of Aß25-35-injected mice when compared to control group. These results show the effect of the selective blocker of IA currents Tx3-1 in both short- and long-term memory retention and in memory impairment caused by Aß25-35, reinforcing the role of IA in physiological and pathological memory processes.

Gastric emptying effect by 4-aminopyridine in patients with chronic spinal cord injury.

BACKGROUND: 4-Aminopyridine (4-AP) given to patients with chronic spinal cord injury (SCI) has shown beneficial effects in some somatic and autonomic functions, although patients often develop dyspeptic symptoms. 4-AP is a potassium-channel blocker capable of altering gastro-pyloric functions as demonstrated experimentally. Our objective was to examine the influence of 4-AP treatment on gastric emptying in patients with chronic SCI. METHODS: Gastric emptying was measured by the acetaminophen absorption test in 18 patients (9 with cervical and 9 with thoracic injury), and 9 healthy volunteers. Patients received increasing oral doses, 5 mg day(-1) of oral 4-AP (5-30 mg day(-1)), for 12 weeks. Patients were studied before and at the end of the last week of 4-AP treatment, whereas healthy volunteers (without 4-AP treatment) were studied only once. Whole blood samples of 2.5 mL were drawn at 0 (before 1 g of oral acetaminophen) and at 15, 30, 45, 60, 75, 90, 105 and 120 min postdose. Acetaminophen concentration in plasma was determined by high-pressure liquid chromatography. RESULTS: Treatment with 4-AP significantly delayed stomach emptying in patients with chronic SCI, considering the significant decreasing of acetaminophen absorption (t paired test, p <0.05). This effect did not correlate either to the level or ASIA score of the injury (linear regression correlation analysis, r(2) = 0.003 and 0.015, respectively). No significant differences were observed by comparing data of patients before 4-AP treatment with healthy volunteers. CONCLUSIONS: 4-AP intake in patients with chronic spinal cord injury significantly slowed gastric emptying regardless of level and ASIA score of the injury.

Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GABA-mediated neurotransmission and of ion channels.

Infusion of the K(+) channel blocker 4-aminopyridine in the hippocampus induces the release of glutamate, as well as seizures and neurodegeneration. Since an imbalance between excitation and inhibition, as well as alterations of ion channels, may be involved in these effects of 4-aminopyridine, we have studied whether they are modified by drugs that block glutamatergic transmission or ion channels, or drugs that potentiate GABA-mediated transmission. The drugs were administered to anesthetized rats subjected to intrahippocampal infusion of 4-aminopyridine through microdialysis probes, with simultaneous collection of dialysis perfusates and recording of the electroencephalogram, and subsequent histological analysis. Ionotropic glutamate receptor antagonists clearly diminished the intensity of seizures and prevented the neuronal damage, but did not alter substantially the enhancement of extracellular glutamate induced by 4-aminopyridine. None of the drugs facilitating GABA-mediated transmission, including uptake blockers, GABA-transaminase inhibitors and agonists of the A-type receptor, was able to reduce the glutamate release, seizures or neuronal damage produced by 4-aminopyridine. In contrast, nipecotate, which notably increased extracellular levels of the amino acid, potentiated the intensity of seizures and the neurodegeneration. GABA(A) receptor antagonists partially reduced the extracellular accumulation of glutamate induced by 4-aminopyridine, but did not exert any protective action. Tetrodotoxin largely prevented the increase of extracellular glutamate, the electroencephalographic epileptic discharges and the neuronal death in the CA1 and CA3 hippocampal regions. Valproate and carbamazepine, also Na(+) channel blockers that possess general anticonvulsant action, failed to modify the three effects of 4-aminopyridine studied. The N-type Ca(2+) channel blocker omega-conotoxin, the K(+) channel opener diazoxide, and the non-specific ion channel blocker riluzole diminished the enhancement of extracellular glutamate and slightly protected against the neurodegeneration. However, the two former compounds did not antagonize the 4-aminopyridine-induced epileptiform discharges, and riluzole instead markedly increased the intensity and duration of the disharges. Moreover, at the highest dose tested (8mg/kg, i.p.), riluzole caused a 75% mortality of the rats. We conclude that 4-aminopyridine stimulates the release of glutamate from nerve endings and that the resultant augmented extracellular glutamate is directly related to the neurodegeneration and is involved in the generation of epileptiform discharges through the concomitant overactivation of glutamate receptors. Under these conditions, a facilitated GABA-mediated transmission may paradoxically boost neuronal hyperexcitation. Riluzole, a drug used to treat amyotrophic lateral sclerosis, seems to be toxic when combined with neuronal hyperexcitation.

Release and uptake of glutamate as related to excitotoxicity.

It has been established that neurons exposed to high concentrations of glutamate or other excitatory amino acids degenerate and die. Neuronal damage appears to be due to the activation of different types of glutamate receptors, among which the ionotropic N-methyl-D-aspartate (NMDA) type seems particularly involved, since its channel is permeable to Ca2+ and an increase in the cytoplasmic concentration of this cation promotes a chain of events leading to cell death. The mechanism of such glutamate receptor-mediated neurodegeneration has been defined as excitotoxicity, and several pieces of evidence suggest that this mechanism might contribute to the neuronal death associated with certain neurological disorders, such as ischemia, cerebral trauma and some chronic neurodegenerative diseases. A relevant question is whether the origin of endogenous extracellular glutamate is important for the induction of excitotoxicity. An excess of glutamate release, or a deficiency in its clearance from the synaptic cleft, which depends mainly on its transport by high affinity carriers, are potential sources for the accumulation of extracellular glutamate. In the present article some experimental results from our laboratory, aimed at obtaining information on this question, are reviewed. These experiments include the use of 4-aminopyridine, a convulsant drug that enhances the release of glutamate, and of some inhibitors of glutamate transport, in vivo and in neuronal cell cultures. The results obtained indicate that an increase of endogenous extracellular glutamate due to these procedures is not sufficient to induce neuronal death, at least under the experimental conditions used.

Release and uptake of glutamate as related to excitatoxicity

It has been established that neurons exposed to high concentrations of glutamate or other excitatory amino acids degenerate and die. Neuronal damage appears to be due to the activation of different types of glutamate receptors, among which the ionotropic N-methyl-D-aspartate (NMDA) type seems particularly involved, since its channel is permeable to Ca2+ and an increase in the cytoplasmic concentration of this cation promotes a chain of events leading to cell death. The mechanism of such glutamate receptor-mediated neurodegeneration has been defined as excitotoxicity, and several pieces of evidence suggest that this mechanism might contribute to the neuronal death associated with certain neurological disorders, such as ischemia, cerebral trauma and some chronic neurodegenerative diseases. A relevant question is whether the origin of endogenous extracellular glutamate is important for the induction of excitotoxicity. An excess of glutamate release, or a deficiency in its clearance from the synaptic cleft, which depends mainly on its transport by high affinity carriers, are potential sources for the accumulation of extracellular glutamate. In the present article some experimental results from our laboratory, aimed at obtaining information on this question, are reviewed. These experiments include the use of 4-aminopyridine, a convulsant drug that enhances the release of glutamate, and of some inhibitors of glutamate transport, in vivo and in neuronal cell cultures. The results obtained indicate that an increase of endogenous extracellular glutamate due to these procedures is not sufficient to induce neuronal death, at least under the experimental condition used.

Protection by NMDA receptor antagonists against seizures induced by intracerebral administration of 4-aminopyridine.

The effects of NMDA receptor antagonists on the convulsant action of the administration of 4-aminopyridine in the rat lateral cerebral ventricle (i.c.v. injection) and motor cerebral cortex (i.cx. injection) were studied. 4-Aminopyridine administration in both regions induced various preconvulsive symptoms, such as salivation, tremors, chewing and rearing, followed by continuous clonic convulsions and, only after i.c.v. injection, running fits and generalized tonic convulsions. This behavioral pattern appeared 5-9 min after administration of 4-aminopyridine and persisted for 100-150 min. 4-Aminopyridine also generated epileptiform electroencephalographic (EEG) discharges characterized by isolated spikes, poly-spikes and spike-wave complexes, which began some seconds after administration of the drug and were present for more than 2 h. The NMDA receptor antagonists (+/-)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), (+/-)-2-amino-7-phosphono-heptanoic acid (AP7) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) clearly protected against some of the behavioral alterations induced by i.c.v. 4-aminopyridine, particularly the tonic convulsions, but were less effective against those produced by i.cx. 4-aminopyridine. These antagonists also delayed the appearance of EEG epileptiform discharges, reduced its amplitude, frequency and duration, and blocked their propagation to other cortical regions after i.cx. 4-aminopyridine. These results, together with previous data showing that 4-aminopyridine stimulates the release of glutamate in vivo, suggest that an excessive glutamatergic neurotransmission involving NMDA receptors is implicated in 4-amino-pyridine-induced seizures.