Time- and Region-Specific Variations in Somatostatin Release Following Amygdala Kindling in the Rat

Abstract: Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin-like immunoreactivity (somatostatin-LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham-stimulated controls, (a) in the hippocampus, high-K⁺-evoked somatostatin-LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin-LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin-LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.     

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.     

Effect of Pentylenetetrazole-Induced Kindling on Acetylcholine Release in the Hippocampus of Freely Moving Rats

Abstract: The role of γ-aminobutyric acid (GABA) modulation of septohippocampal cholinergic neurons in kindling was investigated. Hippocampal acetylcholine release was evaluated with the microdialysis technique in freely moving rats either after acute administration of isoniazid (an inhibitor of GABA synthesis) or pentylenetetrazole (PTZ)(a blocker of the GABA_A receptor-associated Cl⁻ channel) or after chronic administration of PTZ. Short-term treatment with PTZ (5–50 mg/kg, i.p.) or isoniazid (150–250 mg/kg, s.c.) increased hippocampal acetylcholine release in a dose-dependent manner. In contrast, the basal concentration of acetylcholine in the dialysate from the hippocampus of rats chronically treated with PTZ (kindled animals) was significantly reduced relative to that of vehicle-treated rats (2.39 ± 0.21 vs. 4.2 ± 0.31 pmol per 20-min sample; p < 0.01). Moreover, the release of acetylcholine was markedly more sensitive to the effect of a challenge injection of PTZ (10 or 20 mg/kg, i.p.) in kindled rats than in naive rats or rats chronically treated with vehicle. Abecarnil, a selective benzodiazepine receptor agonist with marked anticonvulsant activity, was administered together with chronic PTZ to evaluate whether persistent activation of GABA_A receptors and suppression of seizures during kindling might affect the sensitivity of septohippocampal cholinergic neurons to a challenge dose of PTZ. Abecarnil (1 mg/kg, i.p.) administered 40 min before each PTZ injection neither antagonized the decrease in basal acetylcholine release (2.26 ± 0.19 pmol per 20-min sample) nor prevented the development of kindling. In contrast, abecarnil prevented the chronic PTZ-induced increase in the sensitivity of acetylcholine release to a challenge dose of PTZ. These results provide novel in vivo data concerning the role of hippocampal acetylcholine function in the development of kindling and potentially in the learning and memory deficits associated with this phenomenon.     

Accumulation of 7S SNARE complexes in hippocampal synaptosomes from chronically kindled rats

Kindling is a model of complex partial epilepsy wherein periodic application of an initially subconvulsive stimulus leads to first limbic and then generalized tonic-clonic seizures. Several laboratories have reported that augmented neurotransmitter release of l-glutamate is associated with the chronically kindled state. Neurotransmitter release requires membrane proteins called SNAREs, which form transmembrane complexes that participate in vesicle docking and are required for membrane fusion. We show here that kindling by entorhinal stimulation is associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus. This increase of 7S SNARE complexes appears to begin early in the kindling process, achieves a peak with full kindling, and remains at this level for at least a month following cessation of further kindling stimuli. The increase is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. It is not seen in animals that receive electroconvulsive seizures, suggesting it is related to the kindling process itself. The duration and focality of increased 7S SNARE complexes with entorhinal kindling suggest that this is an altered molecular process associated with epileptogenesis.     

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.     

Amygdala Kindling Potentiates Seizure-Stimulated Immediate-Early Gene Expression in Rat Cerebral Cortex

Kindling induces long-term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate-early genes (IEGs) c-fos, c-jun, and NGF1-A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c-fos mRNA in cerebral cortex, ECS-stimulated levels of c-fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS-stimulated levels of c-fos mRNA were not significantly increased in stage 2 kindled animals. ECS-stimulated levels of c-jun and NGF1-A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was long-lasting because an acute ECS stimulus significantly elevated levels of c-fos and c-jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS-stimulated levels of c-fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina-specific changes in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Z944, a Novel Selective T-Type Calcium Channel Antagonist Delays the Progression of Seizures in the Amygdala Kindling Model

Temporal lobe epilepsy (TLE) is the most common form of drug resistant epilepsy. Current treatment is symptomatic, suppressing seizures, but has no disease modifying effect on epileptogenesis. We examined the effects of Z944, a potent T-type calcium channel antagonist, as an anti-seizure agent and against the progression of kindling in the amygdala kindling model of TLE. The anti-seizure efficacy of Z944 (5mg/kg, 10mg/kg, 30mg/kg and 100mg/kg) was assessed in fully kindled rats (5 class V seizures) as compared to vehicle, ethosuximide (ETX, 100mg/kg) and carbamazepine (30mg/kg). Each animal received the seven treatments in a randomised manner. Seizure class and duration elicited by six post-drug stimulations was determined. To investigate for effects in delaying the progression of kindling, naive animals received Z944 (30mg/kg), ETX (100mg/kg) or vehicle 30-minutes prior to each kindling stimulation up to a maximum of 30 stimulations, with seizure class and duration recorded after each stimulation. At the completion of drug treatment, Ca_V3.1, Ca_V3.2 and Ca_V3.3 mRNA expression levels were assessed in the hippocampus and amygdala using qPCR. Z944 was not effective at suppressing seizures in fully kindled rats compared to vehicle. Animals receiving Z944 required significantly more stimulations to evoke a class III (p<0.05), IV (p<0.01) or V (p<0.0001) seizure, and to reach a fully kindled state (p<0.01), than animals receiving vehicle. There was no significant difference in the mRNA expression of the T-type Ca²⁺ channels in the hippocampus or amygdala. Our results show that selectively targeting T-type Ca²⁺ channels with Z944 inhibits the progression of amygdala kindling. This could be a potential for a new therapeutic intervention to mitigate the development and progression of epilepsy.     

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.     

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.     

Changes in benzodiazepine receptors in the limbic system following kindling of the olfactory bulb in rats

Repeated electrical stimulations of the olfactory bulb led to the progressive development of a generalized epilepsy (kindling effect). One week after the last stimulation eliciting a stage 5 seizure, diazepam-(3H) binding was studied in olfactory bulb-kindled rats. Numbers of benzodiazepine receptors were increased in kindled olfactory bulb and amygdala. No significant change was observed in hippocampus. This modification could be considered as a response of the inhibitory mechanisms to repeated seizures which is insufficient to counteract the installation of the kindling effect.     

Monophosphoryl Lipid A and Pam3Cys Prevent the Increase in Seizure Susceptibility and Epileptogenesis in Rats Undergoing Traumatic Brain Injury

Five percent of all epilepsy cases are attributed to traumatic brain injury (TBI), which are known as post-traumatic epilepsy (PTE). Finding preventive strategies for PTE is valuable. Remarkable feature of TBI is activation of microglia and subsequent neuroinflammation, which provokes epileptogenesis. The toll-like receptor agonists monophosphoryl lipid A (MPL) and tri-palmitoyl-S-glyceryl-cysteine (Pam3Cys) are safe, well-tolerated and effective adjuvants existing in prophylactic human vaccines. We examined the impact of early injection of MPL and Pam3Cys to rats, on the rate of kindled seizures acquisition following TBI. Rats received a single dose (1 µg/rat) of MPL or Pam3Cys through intracerebroventricular injection. 5 days later, trauma was exerted to temporo-parietal cortex of rats by controlled cortical impact device. After 24 h, traumatic rats underwent amygdala kindling. Brain level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) was also measured in traumatic rats by immunoblotting. Compared to non-traumatic (sham-operated) rats, traumatic rats showed three times lower seizure threshold (133?±?5 µA vs. 416.3?±?16 µA, p?<?0.001); about three times less number of stimuli to become kindled (5?±?1 vs. 14?±?2, p?<?0.01); longer duration of kindled seizure parameters including entire seizure behavior, generalized seizures, and afterdischarges (p?<?0.001); and a two times increase in the TNF-α level. MPL and Pam3Cys did not change kindling rate and the seizure parameters in sham-operated rats. The MPL- and Pam3Cys-pretreated traumatic rats displayed seizure threshold, speed of kindling, and duration of kindled seizure parameters, similar to the non-traumatic rats. Pretreatment by MPL and Pam3Cys prevented the increase in TNF-α level by trauma. Given that MPL and Pam3Cys currently have clinical use as well-tolerated vaccines with reliable safety, they have the potential to be used in prevention of PTE.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

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.     

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.     

Amygdala adenosine A1 receptors have no anticonvulsant effect on piriform cortex-kindled seizures in rat

Adenosine is an endogenous anticonvulsant that exerts its effects through A1 receptors. As the piriform/amygdala is a critical circuit for limbic seizure propagation, in this study, the role of basolateral amygdala A1 receptors on piriform cortex (PC)-kindled seizures was investigated. Rats were kindled by daily electrical stimulation of PC. In fully kindled animals, bilateral intra-amygdala N6-cyclohexyladenosine (CHA; 10-500 micromol/L, a selective A1 receptor agonist) had no effect on kindled-seizure parameters. However, bilateral intra-amygdala 2% lidocaine (reversal neuronal inhibitor) reduced the kindled seizure severity. There was significant increase in stage 4 latency and decrease in stage 5 duration. Bilateral lesion of basolateral amygdala of kindled animals (by electrical DC current) reduced the kindled seizure severity more dramatically. Our results showed afterdischarge duration, stage 5 duration, and seizure duration were decreased and stage 4 latency increased significantly. In addition, daily intra-amygdala CHA had no significant effect on PC kindling acquisition. Therefore, it may be concluded that although the basolateral amygdala neuronal activity has a critical role in the propagation of epileptic seizures from PC, the amygdala A1 receptors have no role in this regard. On the other hand, amygdala A1 receptors have no anticonvulsant or antiepileptogenic effect on PC-kindled seizures.     

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.     

Variations of Rat Brain Calmodulin Content in Dark and Light Phases: Effect of Pentylenetetrazol-Induced Kindling

Calmodulin (CaM) through activation of CaM-kinase II may be involved in the molecular mechanisms underlying the epileptogenic processes. Some evidence suggests that kindling responses change across the day-night cycle. In order to test if kindling stimulation modifies CaM content, we measured CaM concentrations in amygdala, hippocampus and hypothalamus obtained from control and kindled rats during light and darkness. Male Wistar rats (250–300 g), were injected i.p. with Pentylenetetrazol (PTZ) (35 mg/kg/24 h). Once chemical kindling was established, rats were sacrificed by decapitation at 10:30 a.m. and 01:30 a.m. The brains were obtained, and the amygdala, hippocampus and hypothalamus dissected. CaM content was measured in the cytosol and membrane fractions by radioimmunoassay. We found a significant increase in CaM content in cytosol and membrane fractions of both control and kindled rats during the dark phase. No significant differences in CaM concentrations were observed between control and experimental rats, whether during the light or the dark phase. The data suggest a well defined photoperiodic variation in CaM concentrations in limbic structures, despite the neuronal excitability produced by kindling. In addition, the observed CaM increases during the dark time may be related to a protective mechanism against enhanced sensitivity to seizures observed during the night.     

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.     

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.