Protein kinase inhibitor as a potential candidate for epilepsy treatment.

PURPOSE: Effects of the "VID-82925" kinase inhibitor molecule were investigated both during the developing phase as well as during the stable phase of the focus with spontaneous recurrent seizures using the 4-AP-induced in vivo epilepsy model in anesthetized rats. METHODS: In electrophysiologic experiments, VID-82925 (0.85 mg/kg) was injected intravenously either before the induction (pretreatment) or after the development of the stable focus (treatment). Reference drugs carbamazepine (4.8 mg/kg) and levetiracetam (50 mg/kg) were employed using the same experimental paradigm. The antiepileptic effect of VID-82925 was also compared to those of the broad-spectrum gap junction channel blocker carbenoxolone (10 mm). KEY FINDINGS: Pretreatment with VID-82925 revealed an antiepileptogenic effect as it suppressed significantly the manifestation of the epileptiform activity not only during the developing phase, but also for a considerable long period during the stable phase of the focus. The current data do not allow us to differentiate an antiictal treatment effect from an antiepileptogenic effect of the compound during the stable phase of the focus. Treatment with VID-82925 was also effective against ictogenesis during the stable phase of the focus. Pretreatment with levetiracetam failed to exert any antiepileptogenic effect. The antiepileptic effects of VID-82925 and of the reference drugs on the epileptiform activity of the stable focus were comparable in intensity; however, the effect of VID-82925 was 2-3 times longer. The effects of VID-82925 and of carbenoxolone overlapped one another to some extent, suggesting that VID-82925 may exert its effects at least partially through blocking of gap junctional communication. SIGNIFICANCE: Our results indicate that inhibition of protein kinases may also provide an effective strategy for the development of a drug that is not only antiepileptic but also depresses the course of epileptogenesis.

Effects of 3-aminopyridine-induced seizures on platelet eicosanoid synthesis.

We investigated the influence of recurrent epileptic seizures on the arachidonic acid (AA) cascade in platelets and brain microvessels, using [(14)C]AA as a tracer substrate and chromatographic determination. The recurrent epileptic seizures of male Wistar rats were induced every second day with 3-aminopyridine (3-AP, 25 mg/kg ip) for two weeks. In the chronic 3-AP model, the earlier epileptic insults resulted in a decreased incidence of limbic seizures and higher survival rate at later administration of 3-AP. After 3-AP treatment, the formation of lipoxygenase products was unchanged, but the total amount of cyclooxygenase (COX) metabolites was decreased both in platelets and brain microvessels. The reduction in COX-mediated eicosanoid synthesis after recurrent seizures was due to the decreased synthesis of vasodilator and vasoconstrictor COX metabolites. In platelets, the 3-AP-treatment reduced the synthesis of vasodilator prostacyclin (PGI(2)), prostaglandin E(2) (PGE(2)) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT), while the synthesis of prostaglandin D(2) (PGD(2)) remained unchanged. In isolated brain capillaries, the PGD(2), PGE(2) and 12-HHT synthesis was decreased after recurrent seizures. As for the vasoconstrictor COX metabolites, both platelets and brain microvessels synthesized significantly lesser amount of prostaglandin F(2alpha) (PGF(2alpha)) and thromboxane A(2) (TxA(2)) upon 3-AP administration. Our results indicate that platelets and isolated brain capillaries synthesize significantly lesser amount of COX metabolites after chronic 3-AP treatment. The decreased conversion of AA into different COX products may play a role in the neuroprotective/preconditional adaptation of the brain against subsequent seizures.

Exaggeration of epileptic-like patterns by nicotine receptor activation during the GABA withdrawal syndrome.

To understand how nicotinic cholinergic receptors may participate in epileptic seizures, we tested the effects of nicotine and of the competitive nicotinic antagonists dihydro-beta-erythroidine and alpha-bungarotoxin on synaptic paroxysmal depolarization shifts (PDSs) and intrinsic bursts of action potentials recorded in slices from rats presenting a cortical status epilepticus. This model named GABA-withdrawal syndrome (GWS) appears consecutive to the interruption of a prolonged intracortical GABA infusion. Effects of both nicotinic antagonists suggest a distinct involvement of alpha4-beta2 and alpha7 subunits in shaping individual PDSs and patterning repetitive bursts. On one hand, in GWS rats, an increase of PDS latency and prolongation of PDS and bursts were induced by nicotine and reduced by dihydro-beta-erythroidine, but not by alpha-bungarotoxin. The K+ blocker tetraethylammonium also increased duration without changing latency. Thus, dihydro-beta-erythroidine-sensitive receptors exert distinct controls on the presynaptic generation of PDS and on the process which terminates PDSs and bursts. On the other hand, alpha-bungarotoxin depolarized neurons and generated rhythmic discharges of clustered bursts. Clustered bursts were also observed in slices obtained from GWS rats treated with the acetylcholinesterase inhibitor eserine. We suggest that both dihydro-beta-erythroidine and alpha-bungarotoxin-sensitive sites control paroxysmic activities in GWS and could be involved in some human and animal epilepsies presenting mutations of nicotinic cholinergic receptors.

Seizure, neurotransmitter release, and gene expression are closely related in the striatum of 4-aminopyridine-treated rats.

The present experiments aimed to compare the length of seizure activity with the time-related increase of transmitter release and the induction of c-fos gene expression in the striatum of the rat. Anesthetized Wistar rats were intraperitoneally treated with 7 mg/kg 4-aminopyridine, and the transmitter levels in the striatum were measured by means of in vivo microdialysis, 30, 60, 90, 120, and 150 min following the treatment. Striatal and neocortical electric activity was monitored with depth and surface electrodes, respectively. The expression level of the c-fos gene was estimated by counting the striatal c-fos-immunostained cell nuclei at the time intervals of the microdialysis. 4-aminopyridine elicited high-frequency seizure discharges in the EEG and significantly increased glutamate, aspartate, GABA, serotonin, noradrenaline, and dopamine levels in the extracellular dialysates. The number of c-fos-stained cell nuclei in the striatum displayed a prolonged increase, showing significantly elevated numbers throughout the experiment. The increase of c-fos expression in time correlated best with the increase of glutamate release, which was also significantly elevated at every sampling time. The GABA release, culminating at 60 min after the seizure onset, correlated best with the cessation of the electrographic seizure. Aspartate, norepinephrine, serotonin, and dopamine displayed transient but significant elevations. We conclude that glutamate plays the essential role (most probably through ionotropic and metabotropic receptors) in the extracellular signaling, which eventually leads to intracellular cascades and c-fos gene expression in the striatum during convulsions.

Effects of continuous low-dose exposure to organic and inorganic mercury during development on epileptogenicity in rats.

The effects of chronic, low-dose fetal and lactational organic (MeHgCl) and inorganic (HgCl2) mercury intoxication on epileptogenicity were investigated and compared in rats. The main observations after both mercury treatments were a facilitated seizure expression and propagation evoked by 4-aminopyridine (4-AP). The seizure susceptibility of the offspring seemed to be in a parallel relation to the mercury concentration in the cortical tissue, which was significantly higher in treated animals as compared to the controls. While MeHgCl enhanced the number of ictal periods, HgCl2 facilitated the duration of seizure discharges in younger animals. HgCl2 intoxication seemed to be more permanent than MeHgCl. Changes in the summated ictal activity--which is an indication of epileptogenicity--were observed in the opposite directions in the two treated groups with increasing age. The amplitudes of seizure discharges were smaller in both mercury-treated groups than in the controls, which could be a consequence of a depressed proliferation of neurons or enhanced cell death in the neocortex. In summary, we observed that adult rats exposed to organic or inorganic mercury chemicals during their embryonic life, are more prone to epilepsy than control rats given only 4-AP.

Concentration- and time-dependent effect of aminooxyacetic acid on cortical epileptogenicity.

In the present electrophysiological study the effect of aminooxyacetic acid (AOAA) on the cortical epileptogenicity, and on the basic electro-cortical activity was investigated in anesthetized rats. AOAA did not induce spontaneous epileptiform discharges but modified the somato-sensory evoked responses and the cortical epileptogenicity (induced by 4-aminopyridine) in the same manner depending on its concentration. AOAA at low concentrations increased the amplitude of evoked responses and the ipsilateral manifestation of epileptiform activity, however, at high concentrations significantly suppressed both the evoked responses and the induction and expression of seizures discharges. The anticonvulsive effect of AOAA was time-dependent (reached its maximum after 2h AOAA pre-treatment) and reversible. AOAA at low concentrations probably increases the efficacy of the NMDA excitatory system and decreases GABA-synthesis, resulting neuronal hyperexcitation. However, AOAA at high concentrations can lead to an effective cortical inhibition through intra- and extracellular accumulation of GABA. The gradual GABA accumulation - up to a certain level - at the synapses could also explain the time-dependency of the anticonvulsive effect of AOAA.

The functional significance of gap junction channels in the epileptogenicity and seizure susceptibility of juvenile rats.

PURPOSE: The functional significance of gap-junction (GJ) channels in seizure susceptibility and induction and maintenance of seizures in the developing rat brain was investigated on the 4-aminopyridine (4-AP) in vivo epilepsy model. METHODS: In electrophysiological experiments, GJs were manipulated with a blocker or opener before induction or at the active epileptic foci between postnatal days 9 and 28 (P9-28). Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 26, 32, 36, and 43 mRNAs at the untreated cortex or epileptic foci. RESULTS: The basic electrocorticogram (ECoG) and Cx messenger RNA (mRNA) expression patterns exhibited characteristic maturation; the 4-AP-induced epileptiform activity correlated well with these changes. Cx mRNA expressions were significantly upregulated around P16 (except for Cx26). The Cx26, 36, and 43 gene inducibility was highest around P16 and then declined significantly. In the youngest animals, the GJ opener induced rhythmic synchronous cortical activity. On maturation, the seizures became focalized and periodic; the discharges accelerated their amplitude and frequency increase. A transient decrease (P13-14) and then increase (P15-16) in seizure susceptibility were followed by a tendency to periodicity and focalization. CONCLUSIONS: The study suggests that GJ communication is involved in rhythm genesis and synchronization of cortical activity and may enhance the epileptogenicity of the developing brain.

Quinine, a blocker of neuronal cx36 channels, suppresses seizure activity in rat neocortex in vivo.

PURPOSE: The selective contribution of neuronal gap junction (GJ) communication via connexin 36 (Cx36) channels to epileptogenesis and to the maintenance and propagation of seizures was investigated in both the primary focus and the mirror focus by using pharmacologic approaches with the 4-aminopyridine in vivo epilepsy model. METHODS: ECoG recording was performed on anesthetized adult rats, in which either quinine, a selective blocker of Cx36, or the broad-spectrum GJ blockers carbenoxolone and octanol were applied locally, before the induction or at already active epileptic foci. RESULTS: The blockade of Cx36 channels by quinine before the induction of epileptiform activity slightly reduced the epileptogenesis. When quinine was applied after 25-30 repetitions of seizures, a new discharge pattern appeared with frequencies >15 Hz at the initiation of seizures. In spite of the increased number of seizures, the summated ictal activity decreased, because of the significant reduction in the duration of the seizures. The amplitudes of the seizure discharges of all the patterns decreased, with the exception of those with frequencies of 11-12 Hz. The blockade of Cx36 channels and the global blockade of the GJ channels resulted in qualitatively different modifications in ictogenesis. CONCLUSIONS: The blockade of Cx36 channels at the already active epileptic focus has an anticonvulsive effect and modifies the manifestation of the 1- to 18-Hz seizure discharges. Our findings indicate that the GJ communication via Cx36 channels is differently involved in the synchronization of the activities of the networks generating seizure discharges with different frequencies. Additionally, we conclude that both neuronal and glial GJ communication contribute to the manifestation and propagation of seizures in the adult rat neocortex.

Involvement of gap junctions in the manifestation and control of the duration of seizures in rats in vivo.

PURPOSE: The possible role of gap junctions in the manifestation and control of the duration of seizures was tested on the 4-aminopyridine-induced epilepsy model in rats in vivo, by using electrophysiologic, pharmacologic, and molecular biologic techniques. METHODS; In electrophysiologic experiments, the functional states of the gap junctions were manipulated with a specific blocker (carbenoxolone) or opener (trimethylamine) at the already active focus of adult, anesthetized rats, 60 min after the induction of the first seizure, which was repeated spontaneously thereafter. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 32, 43, and 36 messenger RNAs (mRNAs) prepared from the areas of the already active primary and mirror foci. RESULTS: After repeated seizures, the expression levels of Cx32, Cx43, and Cx36 mRNAs at the epileptic foci were increased significantly. Blockade of the gap junctions with carbenoxolone shortened the duration of seizures and decreased the amplitude of the seizure discharges, whereas their opening with trimethylamine lengthened the duration and increased the amplitude. Secondary epileptogenesis was facilitated when the gap junctions were opened. CONCLUSIONS; Our findings support the idea that, in epileptic foci, the gap junctions are involved in the expression of rhythmic ictal discharges and in the control of the duration and propagation of the individual seizures in vivo.