Audiogenic kindling in WAG/Rij rats: change in behavioral and electrophysiological responses to repetitive short acoustic stimulation

Running and tonic convulsions induced by sound stimulation (audiogenic seizures, AS) are known to be brainstem-dependent, but their repeated induction leads to the recruiting forebrain structures into AS expression manifested by the development of clonic convulsions and cortical epileptic activity (audiogenic kindling). Behavioral and electrophysiological manifestations of audiogenic kindling were studied in AS-prone WAG/Rij rats exhibiting two types of genetically determined generalized seizures: convulsive audiogenic and nonconvulsive absence (spontaneous spike-wave discharges generated by thalamocortical circuits). Twenty three repeated (with 2 days intervals) sound stimulations inducing a short running episode led to a progressive increase in AS duration from 6.2 +/- 0.4 s to 24.7 +/- 2.9 s mainly due to the appearance of additional postrunning facial-forelimb clonic convulsions of increasing duration and severity. Fully kindled (Racine's stage 5) seizures were accompanied by a bilateral slow-potential wave of cortical spreading depression (SD) nonsynaptically propagating to both striata and by a long-term postictal suppression of spontaneous absence seizures. Neither corticostriatal SD, nor the spike-wave discharges suppression were observed after running induced by sound in non-kindled rats or by attenuated (subthreshold for clonus) sound in kindled rats. Subthreshold stimulation of kindled rats provoked postictal high-amplitude spiking in the cortex. It is concluded that the recruitment of the cortex into a kindled AS network triggers a corticostriatal SD which may underlie the postictal inhibition of non-convulsive seizures, which follow the kindled AS.     

Urethane anesthesia blocks the development and expression of kindled seizures

The effect of anesthetic and subanesthetic doses of urethane on the development of amygdala kindled seizures and on the expression of previously kindled seizures was studied in hooded rats. An anesthetic dose of urethane (1.5 g/kg) almost completely eliminated evoked afterdischarge and completely eliminated convulsive behavior in both groups. It also eliminated the seizure response to pentylenetetrazol. Subanesthetic doses of urethane (0.25 and 0.5 g/kg) strongly attenuated the expression of previously kindled seizures. These results suggest that urethane may not be an appropriate anesthetic for the study of epileptiform phenomena.     

Amygdala kindled seizure stage is related to altered benzodiazepine binding site density

Benzodiazepine receptor binding was examined in rats at 3 stages of amygdaloid kindling (i.e., initial afterdischarge, Stage 3 and Stage 5) immediately or 24 hr after seizure. 3H-diazepam binding site density (Bmax) was significantly increased 24 hr after Stage 3 and Stage 5 kindled seizures in the hippocampus but not in the amygdala. There were no significant differences in the dissociation constants (KD) between kindled and control rats at any time point examined for either brain region. These results demonstrate that changes in benzodiazepine binding are observed with partial kindled seizures (i.e., Stage 3), indicating that generalized seizures are not prerequisite to increased benzodiazepine receptor site density.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis

Abstract: Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 ± 2.1 days (mean ± SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40–60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1–6), dynorphin-A (1–8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.     

Influence of cerebro-spinal fluid of picrotoxin kindled rats on seizure susceptibility and locomotor activity of recipients.

Repeated picrotoxin administration (ip) in subthreshold doses in rats resulted in kindling of generalized seizures. Decrease of locomotor activity in kindled rats occurred in interictal periods. Intra-cerebroventricular microinjection to intact recipients of cerebrospinal fluid (CSF) of kindled but not intact rats or those after acute picrotoxin-induced convulsions, induced a decrease of locomotor activity and severity of acute picrotoxin induced seizures. These effects of CSF were blocked by naloxone pretreatment and were absent after injection of CSF to which protease inhibitors were not added. It is concluded that the release of endogenous opioid peptide substance(s) takes place in CSF of kindled animals which cause the interictal decrease of locomotor activity and may play the role of endogenous anticonvulsive factors controlling epileptic activity induction.     

Failure to kindle seizures after repeated intracerebral administration of arginine vasopressin

In an attempt to kindle seizures with arginine-vasopressin (AVP), we injected AVP into the amygdala or hippocampus of rats. Although behavioral and electrographic alterations were sometimes observed, seizures failed to develop, even in rats that had previously been kindled with electrical stimulation. This and previous failures to kindle seizures by intraventricular injections of AVP call into question the possibility of AVP kindling.     

Modulation of leukotriene D4 attenuates the development of seizures in mice

The present study has been designed to pharmacologically investigate the effect of Montelukast sodium, a leukotriene D₄ receptor antagonist, and 1,2,3,4, tetrahydroisoquinoline, a leukotriene D₄ synthetic pathway inhibitor, on the pathophysiological progression of seizures using mouse models of kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 d was used to elicit chemically induced kindled seizure activity in mice. In a separate set of groups, fifty consecutive electroshocks were delivered to mice using corneal electrodes with continuously increasing intensity with an inter-shock interval of 40 s. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every twenty minutes until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration and electroshock induced the development of severe form of kindled seizures in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, Montelukast sodium, a leukotriene D₄ receptor antagonist, as well as 1,2,3,4, tetrahydroisoquinoline, a leukotriene D₄ synthetic pathway inhibitor, markedly and dose dependently suppressed the development of kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore, leukotriene D₄ may be implicated in the pathogenesis of seizures.     

Involvement of nitric oxide in pentylenetetrazole-induced kindling in rats

We investigated the role of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) in the pentylenetetrazole (PTZ)-induced kindling in rats. Seizures were induced by single administration of PTZ, which was associated with an increase in levels of NO metabolites (NOx) in the hippocampus. Pretreatment with a neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), diminished the PTZ-induced increase in NOx levels without affecting the seizure intensity. Repeated administration of PTZ produced a gradual increase in the seizure intensity, leading to the development of kindling. In the kindled rats, PTZ at a dose of 40 mg/kg increased NOx levels in the hippocampus, whereas it had no effect in control animals. Cotreatment of 7-NI with PTZ blocked the development of kindling and attenuated the PTZ-induced increase in NOx levels. A significant increase in BDNF levels was observed in the hippocampus of the kindled rats, which returned to the control levels following seizures induced by PTZ. 7-NI reduced the hippocampal BDNF levels in control rats and suppressed the increase of BDNF levels in the kindled rats. Our findings suggest that NO plays a role in the development of PTZ-induced kindling and that BDNF may contribute to the NO-dependent plastic changes in neuronal excitability.     

Neurotransmitters and receptor binding in amygdaloid kindled rats: Serotonergic and noradrenergic modulatory effects

No significant difference could be found between 15 amygdaloid kindled rats and 15 implanted but non-stimulated control rats with respect to 10 receptor binding assays carried-out in various brain regions. In a further group of 33 kindled rats neurotransmitter agonists and antagonists were tested for their effects on the duration of the clonic forepaw component. ACh and DA agonists or antagonists had no significant effects. However, the serotonin antagonists and clonidine reduced, whereas 5HTP increased, the duration of clonus. Results are interpreted as an opposing modulatory effect of serotonin and noradrenaline in kindled seizures.     

Effect of chronic para-chlorophenylalanine treatment on convulsive-seizure reactions

The effect of chronic para-chlorphenylalanine (PCPA) treatment was investigated in two different seizure models: the pentylenetetrazole (PTX) seizure model in rats and the kindled seizures from rabbit amygdala. Chronic PCPA treatment (21 days) in male albino rats caused a progressive decrease in the 5-hydroxytryptamine (5-HT) brain level between the 1st and the 7th day of PCPA administration. Then the 5-HT level remained low until the end of the experiment. On the background of the low 5-HT level there occurred changes in PTZ convulsive reactions: after the 3rd day of PCPA treatment the convulsive-seizure reactivity was significantly increased and after the 7th, 14th and 21st day the increased seizure reactivity performed only as a tendency, though the 5-HT level was still low. Chronic PCPA treatment (16 days) of rabbits delayed the development of the behavioural kindled seizures. This treatment also reduced the duration of bioelectrical seizures until the 8th day of treatment, especially in the motor cortex. The observed different effect of the chronic PCPA treatment in both seizure models: pentylenetetrazole in rats and kindling in rabbits might be explained by essential differences in the origin and mechanisms of development of the two seizure models.     

dTyr-D-Phe3 (Pro-D-Phe-Pro-Gly) interacts specifically with amygdaloid-kindled seizures and is capable of preventing the learning deficit occurring after kindling.

In amygdala-kindled rats a deficit in learning brightness discrimination was found. We concluded that this result provides a reliable basis for creating an animal model of cognitive dysfunctions in epileptics. Recently, a des-tyrosine D-amino acid substituted derivative of bovine beta-casein(1-5) was reported to exhibit anticonvulsant as well as antidepressant activity. Therefore, we tested the effect of this peptide on kindled seizures and the learning deficit after kindling. It was found that the peptide suppressed the duration of seizures whereas seizure severity was not influenced. Furthermore, the learning performance of peptide-treated rats was significantly higher than that of kindled controls.     

The development of kindled seizures is accelerated in the genetically epilepsy-prone rat

The kindling phenomenon was examined in genetically epilepsy-prone (GEPR) and non-epileptic control Sprague-Dawley rats. Kindling stimulations were administered three times a day until each rat had exhibited three Class 5 kindled motor seizures. The mean total number of kindling stimulations required for each experimental group to exhibit three motor seizures of each motor seizure class was determined. The results indicated that the early stage of kindling development was accelerated significantly in both the GEPR-3 and GEPR-9 rats, compared to non-epileptic control rats. Later stages of kindling development were accelerated in GEPR-9 but not GEPR-3 rats. Thus a differential acceleration of kindling development was exhibited by GEPR-3 and GEPR-9 rats. The results suggest the possibility that some brain region(s) involved in the early stages of kindling development may be hyperexcitable in both GEPR-3 and GEPR-9 rats. Other brain region(s) involved with the later stages of kindling development may be more excitable in GEPR-9 rats. These putative alterations may, in part, contribute to the seizure prone state of GEPR rats and the differential seizure responses of GEPR-3 and GEPR-9 rats.     

The role of the opiate mechanisms of the hippocampus and substantia nigra in the behavioral and convulsive disorders in picrotoxin-induced kindling]

It was shown in the experiments on rats that the repeated picrotoxin administration resulted in the kindling of generalized seizures. Generalized convulsions were followed by the development of either postictal depression or explosiveness. The injection of mu-opiate agonist met-enkephalin into hippocampus of kindled rats resulted in the increase in the severity of seizure reactions which were induced by picrotoxin and also in the increase in the number of animals with postictal explosiveness. The injection of dynorphin-A-1-13 (kappa-opiate agonist) into substantia nigra reticulata induced the locomotor depression which was like one in postictal period and resulted in the decrease of picrotoxin-induced seizures severity. It was concluded that mu-opiate system of hippocampus took part in the formation of generator of pathologically enhanced excitation in the structure during kindling and the development of seizure syndrome, providing also the postictal explosiveness. Kappa-opiate system of substantia nigra plays an important role in the activation of the antiepileptic system, limitation of seizures and the development of postictal depression.     

Effect of brain extracts from rats subjected to korazol kindling on generalized epileptic activity

The pharmacological kindling was induced in rats by corazol repeated injections in subthreshold doses. The peptide-containing fraction was emitted from animal brains by the help of hot acetic acid on the stage of generalized clonic-tonic seizures development. Intraperitoneal injection of brain extracts of kindled rats significantly increased corazol and picrotoxin induced seizure severity in mice. The effect was removed by preliminary injection of naloxone or by preventive incubation of extracts with pronase. Intraventricular injection of extracts to intact rats increased the seizure severity which was provoked by corazol and in high doses induced in rats generalized seizure reactions.     

Anticonvulsant effect of A1 but not A2A adenosine receptors of piriform cortex in amygdala-kindled rats

In this study, the effect of A1 and A2A adenosine receptor activity of the piriform cortex (PC) on amygdala-kindled seizures was investigated in rats. Animals were kindled by daily electrical stimulation of the amygdala. In fully kindled rats, N6-cyclohexyladenosine (CHA, a selective A1 agonist), 8-cyclopentyl-1,3-dimethylxanthine (CPT, a selective A1 antagonist), CGS21,680 hydrochloride (CGS, a selective A2A agonist), and ZM241,385 (ZM, a selective A2A antagonist) were microinjected bilaterally into the PC. Rats were stimulated 5 min post-drug microinjection and seizure parameters were measured. Results showed that intra-PC CHA (10 and 100 micromol/L) decreased the duration of both afterdischarge and stage 5 seizure and significantly increased the latency to stage 4 seizure. Intra-PC CPT increased afterdischarge and stage 5 seizure duration at the dose of 20 micromol/L. The anticonvulsant effect of CHA (100 micromol/L) was eliminated by CPT (10 micromol/L) pretreatment. On the other hand, neither intra-PC CGS nor ZM had a significant effect on kindled seizures. These results suggest that activity of A1, but not A2A, receptors of the PC have anticonvulsant effects on kindled seizures elicited from electrical stimulation of the amygdala.     

Muscarinic modulation of GABAergic transmission to neurons in the rat subfornical organ

Cholinergic actions on subfornical organ (SFO) neurons in rat slice preparations were studied by using whole cell voltage- and current-clamp recordings. In the voltage-clamp recordings, carbachol and muscarine decreased the frequency of GABAergic inhibitory postsynaptic currents (IPSCs) in a dose-dependent manner, with no effect on the amplitudes or the time constants of miniature IPSCs. Meanwhile, carbachol did not influence the amplitude of the outward currents induced by GABA. Furthermore, carbachol and muscarine also elicited inward currents in a TTX-containing solution. From the current-voltage relationship, the reversal potential was estimated to be -7.1 mV. These carbachol-induced responses were antagonized by atropine. In the current-clamp recordings, carbachol depolarized the membrane with increased frequency of action potentials. These observations suggest that acetylcholine suppresses GABA release through muscarinic receptors located on the presynaptic terminals. Acetylcholine also directly affects the postsynaptic membrane through muscarinic receptors, by opening nonselective cation channels. A combination of these presynaptic and postsynaptic actions may enhance activation of SFO neurons by acetylcholine.     

Effect of amygdaloid kindling on rat striatal dopamine D1- and D2-receptors

The effect of kindling on dopaminergic (DA) neurotransmission was assessed by measuring dopamine D₁- and D₂-receptor binding in the dorsal and ventral striatum of rats either 2 hours (short-term) or 3–4 weeks (long-term) after the last kindled seizure. Kindling did not have any significant long-term effect on DA D₂-receptor K_d or B_max values in the dorsal or ventral striatum or on DA D₁-receptor parameters in the dorsal striatum. The short-term effect of kindled seizures was to abolish the asymmetry in DA D₂-receptor density observed in the dorsal striatum of control rats. DA D₁-receptor density was also increased in the dorsal striatum contralateral to the kindled amygdala of short-term rats. The short-term effects support the notion that limbic seizures can modify the lateral imbalance of DA activity in the striatum.     

Anticonvulsant effects of magnesium sulfate in hippocampal-kindled rats

The objective of the present study was to determine whether magnesium sulfate has anticonvulsant actions in the hippocampal-kindled rat model of epilepsy. Fully kindled rats received acute intraperitoneal injections of magnesium sulfate (270 mg/kg), phenytoin (20 mg/kg) or saline in random order. Electrical seizure duration, behavioral seizure stage and duration of postictal EEG depression were examined 15, 30 and 60 min after injection. In an additional group of rats, kindled seizures were measured before and after chronic (2 h) intraperitoneal injections of magnesium sulfate versus saline. There was a significant decrease in electrical seizure duration (p<0.01) and behavioral seizure stage (p<0.01) with acute magnesium sulfate injections compared to saline injections. Phenytoin had no statistically significant effects on hippocampal-kindled seizures. Chronic magnesium sulfate treatment significantly reduced behavioral seizure stage at 2, 24, and 48 h postinjection (p<0.05), but did not affect seizure duration. There was a significant time by treatment effect for magnesium sulfate on postictal EEG depression (p<0.01). We conclude that in this model of hippocampal epilepsy-induced (kindled) rats, magnesium sulfate has significant anticonvulsant effects.