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.     

Independent expression of cyclic AMP dependent protein kinases related to cyclic nucleotide systems, during triiodothyronine induced cardiac hypertrophy

Daily injections of subconvulsive amounts of carbamylcholine or muscarine into the L basolateral amygdala of Holzman rats resulted in the progressive development of kindled seizures. Addition of equimolar atropine to carbachol completely prevented development of seizures. Rats kindled with carbachol had full seizures when tested for the first time with muscarine and vice-versa. Kindling persisted after 4 weeks without stimulation and spontaneous seizures were observed. No histological differences existed between carbachol-kindled and carbachol-atropine (non-kindled) rats. These data suggest that a chronic epileptic focus was induced transsynaptically.     

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.     

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.     

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.     

The effect of taurine on kindled seizures in the rat

Recently, it has been reported that taurine, an amino acid with anticonvulsant properties, does not suppress experimental seizures generated by the "kindling" technique. This finding seems somewhat paradoxical since taurine antagonizes other sorts of experimental convulsion and since kindled seizures are easily suppressed by other anticonvulsant drugs. Further tests were therefore conducted during which taurine's anticonvulsant effects were assessed: (1) when kindling stimulation was dropped to near-threshold levels; (2) when cortical as well as limbic kindled foci were stimulated; (3) when developing as well as fully kindled seizures were involved; and (4) when taurine was introduced directly into the ventricles of the brain. Even in these tests which were specifically designed to favour the appearance of anticonvulsant effects, no taurine antagonism of kindled seizures was found.     

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.     

The anticonvulsive action of the intranigral administration of the delta sleep-inducing peptide]

It is shown that injection of delta sleep-inducing peptide (DSIP) into the substantia nigra reticular part (SNrp) suppresses generalized convulsive activity induced in rats by picrotoxin and corazol injection but exerts no influence on the strichnine-induced seizures. The analogous DSIP injection causes the antiepileptic action in rats kindled through picrotoxin injections. The DSIP intranigral anticonvulsant action is blocked by naloxon and enhanced by haloperidol and yohimbin. It can be concluded that DSIP anticonvulsant action may be realized due to activation of SNrp-dependent opioid mechanisms and suppression of dopaminergic ones.     

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.     

Increased Phosphorylation of a Membrane Protein Consequent to Amygdaloid Kindling

The phosphorylation of both particulate and soluble proteins in the amygdala was examined in electrically kindled rats. In animals receiving electrical stimulation in the left amygdala for 5-6 days that displayed electrical after-discharges but no motor seizures, no changes were observed in the phosphorylation of either particulate or soluble proteins. In animals stimulated for 20-21 days where major motor seizures were produced, the phosphorylation of a protein having a molecular weight of 45,000 ( 45K ) was markedly increased. The phosphorylation of this protein was increased in both the right (unstimulated) and left (stimulated) amygdala. Major motor seizures induced by electroconvulsive shocks, however, did not alter phosphorylation of this protein. Phosphorylation of the 45K protein was stimulated by calcium and calmodulin. The 45K protein is a major phosphoprotein of amygdala, representing 3.2% of the total particulate phosphoproteins in control animals and 7.4% in the kindled animals. In the presence of calcium-calmodulin, 16.2% of net protein phosphorylation was accounted for by the 45K protein.     

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.     

The effect of delta sleep-inducing peptide on the convulsive activity in corasol kindling

The influence of intraperitoneal delta-sleep inducing peptide (DSIP) injection (100 micrograms/kg) on the epileptic activity was investigated in the experiments on Wistar rats and (CBA X C57B1/6)F1 mice. The model of chronically developing epileptic activity--the model of pharmacological kindling--was created by daily repeated corasole injections in subconvulsive doses (30 mg/kg). It has been shown that DSIP injection delayed the manifestation of generalized seizures during kindling, led to the suppression of seizure activity and reduced the mortality rate of animals that developed kindled seizures. The antiepileptic effect of DSIP was observed throughout the period of 5 minutes to 24 hours after the injection. Naloxone (2.5 mg/kg) did not change the antiepileptic effect of DSIP.     

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.     

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.     

Role of Endoplasmic Reticulum Stress in the Amygdaloid Kindling Model of Rats

Endoplasmic reticulum (ER) is an organelle responsible for correct folding and sorting of proteins contributing to neurogenesis and neuronal cell death. We used rapid kindling to analyze specific ER stress marker expression underlying focal epileptogenesis. Seven-week-old rats were divided into three groups: sham (n = 6), partially kindled (n = 8), and over-kindled rats (n = 9). Over kindled rats received over100 stimuli. Partially kindled animals had stimuli halted at stage 2. Protein from ipsilateral hippocampus was electrophoresed on SDS–PAGE, followed by hybridization with primary antibodies, anti-KDEL (-Lys-Asp-Glu-coo-), Bcl-2, BDNF (brain-derived neurotrophic factor), CHOP (C/EBP-homolog protein), C/EBP (CCAAT/enhancer-binding protein), NMDA (N-methyl-d-aspartate; -R1 &2A), GluR1 (glutamate receptor), and β-tubulin. Western blotting revealed that the ER stress marker BiP (immunoglobulin heavy chain-binding protein) was markedly increased in both partially- and over-kindled groups. BiP expression was ninefold greater than control in partially kindling while twofold greater than control in over-kindled animals. Although ER stress response was accelerated, CHOP expression, which upregulates when apoptosis signaling is accelerated by ER stress, was suppressed. Bcl-2, which acts as an anti-apoptotic molecule, was upregulated in the over-kindled group. Remarkable elevation of BiP was found in partially kindled animals, but not in over-kindled. Over-kindled rats had spontaneous generalized seizure, while partially kindled ones had only partial seizures. Elevation of markers of ER stress in partial seizures might reflect transfer of discharge to contralateral limbic structures. We observed indications of functional changes and neurogenesis in limbic structure during kindling. Widespread indications of functional changes in several membrane and secreted proteins, including NMDA-R1 & R2A and BDNF, for mossy fiber re-construction on the CA3 area, which are related to protein synthesis in the ER, may be important in epileptogenesis.     

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.     

Core mechanisms in generalized convulsions

A hypothetical model is proposed to account for the generalized convulsions observed in rats. Two versions of the hypothesis are discussed: an earlier, more specific formulation that accounts for kindled convulsions, and a later, general version that applies to generalized convulsions as a whole. Observations leading to the specific formulation of the model included: 1) kindling data that suggested a single downstream center responsible for kindled convulsions; 2) brain-stem stimulation data that indicated that the reticular core of that structure could initiate and maintain generalized convulsive behavior; and 3) spinal hemisection data that indicated that nonspecific systems in the cord could also maintain convulsions, even in the absence of direct input from the brain. The more specific version of the model suggests that kindled convulsions are nonspecific core seizures that occur when self-sustained epileptic activation spreads from forebrain foci to involve descending polysynaptic pathways in the nonspecific core of the brain stem and cord. Observations leading to a more general formulation of the model include the facts that: 1) maximal and submaximal convulsions in a variety of models resemble each other; and 2) they also resemble the maximal and submaximal seizures produced by direct stimulation of the brain stem and cord. The more general formulation of the nonspecific core hypothesis suggests that a wide variety of convulsions in rats may be nonspecific core seizures, differences in tonic-clonic and rostrocaudal configuration being related primarily to differences in the intensity of core activation.     

Cysteamine normalizes cerebral somatostatin level and binding in pentylenetetrazol-kindled rats

Rats were kindled by intraperitoneal injection of pentylenetetrazol (PTZ) (30 mg/Kg) every 48 h. Once kindled, some of the animals received a single injection of cysteamine (200 mg/Kg). Somatostatin-like immunoreactivity (SLI) and 125 I-Tyr11-somatostatin binding were measured in the frontoparietal cortex and hippocampus of the two experimental groups and the control rats. After PTZ kindling the following was observed: 1) SLI content was increased in the two areas; 2) Somatostatin receptor affinity decreased in the frontoparietal cortex and was unaltered in the hippocampus; 3) The number of somatostatin receptors decreased in the hippocampus and was unaltered in the frontoparietal cortex. Cysteamine, an agent which depletes brain somatostatin and suppresses kindled seizures in PTZ-treated rats, reversed the altered SLI levels and binding in these rats.