Development Of Amygdala Kindling Research Articles

Identification of QTLs Involved in the Development of Amygdala Kindling in the Rat

Amygdala kindling is useful for modeling human epilepsy development. It has been known that genetic factors are involved in the development of amygdala kindling. The purpose of this study was to identify the loci that are responsible for the development of amygdala kindling. To achieve this, rat strains from a LEXF/FXLE recombinant inbred (RI) strain panel were used. The phenotypes of amygdala kindling-related parameters for seven RI strains and parental LE/Stm and F344/Stm strains were determined. They included the afterdischarge threshold (ADT), the afterdischarge duration (ADD), and the kindling rate, an incidence of development of kindling. Quantitative trait loci (QTL) analysis was performed to identify linkage relationships between these phenotypes and 1,033 SNP markers. Although no significant differences in pre-kindling ADT and ADD were observed, a significant difference in the kindling rate was found for the LEXF/FXLE RI strain. Two QTLs for the amygdala kindling rate (Agkr1 and Agkr2) were identified on rat chromosome 2. These findings clearly prove the existence of genetic influences that are involved in kindling development and suggest that substantial genetic components contribute to the progression of partial seizures into generalized seizures.

Physical exercise: Potential candidate as complementary therapy for epilepsy

Sir, We read with great interest the review article entitled “Nonpharmacological Treatment of Epilepsy” by Saxena and Nadkarni that has appeared in Ann Indian Acad Neurol.-[1] The authors reported several evidences from benefits of non-pharmacological treatment of epilepsy. They notably highlighted that therapies cited in their manuscript should not be considered as alternatives to antiepileptic medication, but as complementary therapy for epilepsy. We applaud the authors for pursuing this topic, but we are also interested in addressing some goals for this review. A large number of people with epilepsy use alternative therapies along with their conventional medicine. Although the practice of Yoga may also be referred to as exercise, programs of physical exercise (such as endurance, strength, balance and flexibility) have an important role in this picture. It is interesting to note that individuals with epilepsy and health care professionals do not usually include physical exercise programs as complementary therapy (for review see Arida et al.[2]). In accordance with this reasoning, positive effects of exercise to people with epilepsy have been increasingly reported in the literature. Studies have generally shown that physical activity can decrease seizure frequency as well as lead to improved cardiovascular and psychological health in people with epilepsy.[3–6] From an experimental point of view, beneficial actions of exercise in animals with epilepsy have been reported by our research group in the last decades. For instance, physical exercise reduced the amygdala kindling development[7] and the frequency of seizures in the pilocarpine model of epilepsy.[8] Metabolic, electrophysiological and immunohistochemical studies also demonstrated encouraging effects of exercise in rats with epilepsy.[9–11] Considering the influence of physical exercise on brain function and positive impact in seizure control, it seems reasonable to include programs of exercise as complementary and alternative medicine (CAM) in epilepsy. Epileptologists should bear in mind the therapeutic use of regular physical exercise when advising the patient to CAM. They should stimulate patients to the practice of regular physical activities not only for seizure control but also for amelioration of general health. Overall, we would like to express our congratulations to Saxena and Nadkarni[1] on their interesting paper and emphasize the possible role of exercise interference as complementary non-pharmacological treatment of epilepsy.

Physical exercise program reverts the effects of pinealectomy on the amygdala kindling development

Several studies have demonstrated the anticonvulsant effect of melatonin. In view of the positive effects of physical exercise in epilepsy, this study analyzed the influence of physical exercise program on the amygdala kindling development in pinealectomized rats. Animals were divided into six groups: pinealectomized rats (PX), sham rats (SHAM), control rats (CTL), pinealectomized rats submitted to an aerobic exercise program (PX ATL), sham rats submitted to an aerobic exercise program (SHAM ATL) and control rats submitted to an aerobic exercise program (CTL ATL). The stimulus parameters consisted of 60 Hz frequency, diphasic square pulses of 1 ms duration applied for 2 s. The mean number of stimulations and the after-discharge (AD) duration for each stage of kindling were similar among CTL and SHAM animals. PX animals showed particular characteristics during kindling development. They did not present stage 1 and spent a shorter time in stage 2 in relation to the CTL and SHAM animals. Consequently, the AD duration and number of stimulations required to reach stage 5 was lower for the PX group when compared to the CTL and SHAM groups. Concerning the exercising groups, CTL ATL and SHAM ATL animals spent a higher time in stage 1 compared to CTL and SHAM groups. Thus, CTL ATL animals also presented a higher number of stimulations in stage 5 compared to CTL animals. The stage 1 not observed in PX animals was present in PX ATL. Consequently, the number of stimulations required to reach stage 5 was statistically higher for the PX ATL group in relation to the PX group. Our results demonstrate that the acceleration in the kindling development of pinealectomized animals can be reverted by physical exercise.

Influence of pinealectomy on the amygdala kindling development in rats

A considerable number of studies have demonstrated the anticonvulsant effect of melatonin. The present study examines the influence of pinealectomy on the amygdala kindling development in rats. Animals were divided into three groups: Pinealectomized rats (PNT) Sham rats (SHAM) and Control rats (CTL). The mean number of stimulations and the afterdischarge (AD) duration for each stage of kindling were similar among CTL and SHAM animals. Conversely, PNT animals showed particular characteristics during kindling development. They did not present stage 1 and spent a shorter time in stage 2 in relation to the CTL and SHAM animals. Consequently, the number of stimulations required to reach stage 5 was lower for the PNT group when compared to the CTL and SHAM groups. Besides, a longer AD duration during stage 5 in the PNT group was also observed. The present findings indicate that the pineal gland removal exert a significant influence on amygdala kindling development suggesting that melatonin holds potential interest in experimental therapy for epilepsy.

Long-term cosequences of intrahippocampal kainate injection in the Proechimys guyannensis rodent

The Proechimys guyannensis (PG), a spiny rodent specie living in the Amazonian region has been recently studied as an animal model of anti-convulsant mechanisms. The PG was found to be resistant to the administration of the muscarinic cholinergic agonist pilocarpine or the amygdala kindling development. This study examined the susceptibility of this animal species to the intrahippocampal kainic acid (KA) injection. Electrographic, behavioral and neuropathological changes induced by intrahippocampal KA injections were analyzed. PG showed to be extremely sensitive to the acute effects of the KA injection. Although the EEG findings in PG rodents were similar to those typically obtained in Wistar rats the pattern of electrographic activity in PG animals was longer than in Wistar rats. Neuropathological examinations of PG brains that survived KA-induced SE revealed severe cell loss in CA1/CA3 areas of the hippocampus, an extensive cell dispersion in the hilus of DG at the injected site with mossy fiber sprouting in the dentate gyrus supragranular layer. None of PG animals presented spontaneous seizures during the 120 days of observation. These findings confirm our previous observation on the resistance of this animal specie to experimental models of limbic epilepsy.

Ictogenesis

Ictogenesis

Calbindin-D 28k content and firing pattern of hippocampal granule cells in amygdala-kindled rats: a perforated patch-clamp study

The dentate gyrus is believed to play an important pathophysiological role during experimentally induced kindling. In this study, we investigated whether an altered content of the calcium binding protein calbindin-D 28k or an increased intrinsic excitability of hippocampal granule cells contribute to the induction of the kindling phenomenon. We determined the firing pattern of granule cells in hippocampal slices using perforated patch-clamp recordings in current clamp mode. The expression of calbindin-D 28k and glutamic acid decarboxylase (GAD 67) by granule cells was analyzed immunohistochemically. Rats developed secondarily generalized limbic seizures within ∼11 days of twice-daily stimulation of the amygdala. As reported for other kindling paradigms, this protocol induced a clear up-regulation of GAD 67 in granule cells, indicating their involvement in the induced neuronal activity. However, when comparing kindled and control rats, we could not detect any differences in intrinsic excitability: Firing frequency, after-hyperpolarisations, action potentials, input resistance and membrane potentials were nearly identical between both groups. Furthermore, we did not observe any differences in the calbindin-D 28k immunoreactivity between groups. In every slice, virtually all granule cells were found to be strongly calbindin-D 28k positive, and there was no apparent reduction in the general level of calbindin-D 28k expression. We conclude that changes in intrinsic membrane properties or in the calbindin-D 28k content of granule cells are not necessary for the development of amygdala kindling.

Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models

Neuropeptide Y (NPY) administered exogenously is anticonvulsant, and, NPY null mutant mice are more susceptible to kainate-induced seizures. In order to better understand the potential role of NPY in epileptogenesis, the present studies investigated the development of amygdala kindling, post-kindling seizure thresholds, and anticonvulsant effects of carbamazepine and levetiracetam in 129S6/SvEv NPY(+/+) and NPY(−/−) mice. In addition, susceptibility to pilocarpine- and kainate-induced seizures was compared in NPY(+/+) and (−/−) mice. The rate of amygdala kindling development did not differ in the NPY(−/−) and NPY(+/+) mice either when kindling stimuli were presented once daily for at least 20 days, or, 12 times daily for 2 days. However, during kindling development, the NPY(−/−) mice had higher seizure severity scores and longer afterdischarge durations than the NPY(+/+) mice. Post-kindling, the NPY(−/−) mice had markedly lower afterdischarge thresholds and longer afterdischarge durations than NPY (+/+) mice. Carbamazepine and levetiracetam increased the seizure thresholds of both NPY (−/−) and (+/+) mice. In addition, NPY (−/−) mice had lower thresholds for both kainate- and pilocarpine-induced seizures. The present results in amygdala kindling and chemical seizure models suggest that NPY may play a more prominent role in determining seizure thresholds and severity of seizures than in events leading to epileptogenesis. In addition, a lack of NPY does not appear to confer drug-resistance in that carbamazepine and levetiracetam were anticonvulsant in both wild type (WT) and NPY null mutant mice.

Lipopolysaccharide retards development of amygdala kindling but does not affect fully-kindled seizures in rats

Seizures are common sequel to brain insults in cases such as stroke, trauma and infection where there is a certain neuroinflammation. Intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) induces an inflammatory state in brain that is used as a model of neuroinflammation. We studied the effect of LPS (0.25 and 2.5 μg/rat, i.c.v.) on development of electrical kindling of the amygdala and on fully-kindled seizures. LPS, at the doses used, had no effect on fully-kindled seizures and afterdischarge (AD) duration at 0.5, 2 or 4 h after administration. However, daily injection of LPS (2.5 μg/rat) retarded acquisition of kindled behavioral seizures. This antiepileptogenic effect could be due to the release of inflammatory mediators from microglia and the related morphological and functional changes in synaptic neurotransmission.

Temporal and spacial changes of highly polysialylated neural cell adhesion molecule immunoreactivity in amygdala kindling development

To investigate the migration of neural stem cells as well as neural plastic changes in epileptic brain, spaciotemporal expression of immunoreactive highly polysialylated neural cell adhesion molecule (PSA– NCAM) was examined in amygdala kindling development of rat. The neural migration and synaptic remodeling detected with PSA–NCAM staining occurred in dentate gyrus of hippocampus, subventricular zone and pyriform cortex with amygdaloid kindling in generalized seizure but not in partial seizure. Although PSA–NCAM positive dendrite in dentate gyrus was minimally found in the control brain, it extended slightly in animals with partial seizure, and greatly toward the molecular layer with generalized seizure. Thus, the migration of neural stem cells as well as neural plastic changes were spacially and temporally different between brain regions depending on different kindling stages. These changes may mainly contribute to the reorganization of neural network in epileptic brain.

Temporal and spacial changes of BrdU immunoreactivity in amygdala kindling development

To investigate the proliferation of neural stem cells (NSC) in epileptic brain, spaciotemporal expression of immunoreactive bromodeoxyuridine (BrdU) was examined in kindling development of rat. Amygdaloid kindling in partial seizure (PS) was effective in proliferation of NSC detected with BrdU-labeling in subventricular zone (SVZ), but not in dentate gyrus (DG). In SVZ, however, the BrdU-labeling cells decreased at stage generalized seizure (GS). These facts indicate that proliferation of NSC increased with PS and decreased with more severe seizures of GS in SVZ, that such a proliferation did not occur in DG with PS or GS. Thus, the proliferation of NSC was spacially and temporally different between brain regions depending on different kindling stages. [Neurol Res 2002; 24: 593-596]

Basal forebrain neurons suppress amygdala kindling via cortical but not hippocampal cholinergic projections in rats.

Intraventricular administration of the immunotoxin 192 IgG-saporin in rats has been shown to cause a selective loss of cholinergic afferents to the hippocampus and cortical areas, and to facilitate seizure development in hippocampal kindling. Here we demonstrate that this lesion also accelerates seizure progression when kindling is induced by electrical stimulations in the amygdala. However, whereas intraventricular 192 IgG-saporin facilitated the development of the initial stages of hippocampal kindling, the same lesion promoted the late stages of amygdala kindling. To explore the role of various parts of the basal forebrain cholinergic system in amygdala kindling, selective lesions of the cholinergic projections to either hippocampus or cortex were produced by intraparenchymal injections of 192 IgG-saporin into medial septum/vertical limb of the diagonal band or nucleus basalis, respectively. Cholinergic denervation of the cortical regions caused acceleration of amygdala kindling closely resembling that observed after the more widespread lesion induced by intraventricular 192 IgG-saporin. In contrast, removal of the cholinergic input to the hippocampus had no effect on the development of amygdala kindling. These data indicate that basal forebrain cholinergic neurons suppress kindling elicited from amygdala, and that this dampening effect is mediated via cortical but not hippocampal projections.

Suppression of epileptogenesis by modification of N-methyl-D-aspartate receptor subunit composition.

The effects of altered N-methyl-D-aspartate (NMDA) receptor subunit composition on seizure development in kindling epilepsy were assessed in transgenic mice expressing high neuronal levels of NR2D under control of the calcium/calmodulin kinase II alpha subunit (alphaCaMKII) promoter. The NR2D subunit is normally present at very low levels in the mature forebrain. Transgenic mice showed a marked reduction of amygdala kindling development. Spread of epileptic activity was retarded and generalized seizures appeared later in animals overexpressing NR2D compared with wild-type mice. The progressive lengthening of epileptiform activity, which normally occurs in kindling, was also dampened in transgenic animals. We conclude that NMDA receptor subunit composition determines the progression of experimental epilepsy.

Effect of physical exercise on kindling development.

The relationship between epilepsy and exercise is a subject of controversy and needs more investigation. We report a study of the effect of physical activity on the development of amygdala kindling in rats. To analyze the acute and chronic effects of exercise on kindling development, 45 rats were divided randomly into three groups. The first group (acute group, n=15) was submitted to a daily bout of aerobic exercise (40 min running on the treadmill at 20 m/min) and kindling stimulated 1 min post-exercise; the second group (chronic group, n=15) was submitted to an aerobic training program (40 min running on the treadmill at 20 m/min, 7 days per week, for 45 days) and then submitted to the same procedure as the first group (daily bout of aerobic exercise -- 40 min running on the treadmill at 20 m/min and kindling stimulated 1 min post-exercise); and the third group (n=15) served as control. All groups were kindling stimulated until they reached stage 5 of kindling. The number of stimulations required to reach stage 5 was statistically higher for the chronic exercise group when compared to the acute exercise group and the control group. A longer time spent in stage 1 and a shorter after-discharge duration in stage 1 was observed in the acute and chronic exercise groups in relation to the control group. This finding suggests that chronic physical exercise inhibits development of amygdala kindling in rats.

Antiepileptic effects of tiagabine, a selective GABA uptake inhibitor, in the rat kindling model of temporal lobe epilepsy.

We determined the antiepileptic profile of tiagabine (TGB), a selective gamma-aminobutyric acid (GABA) uptake inhibitor, in the rat kindling model of temporal lobe epilepsy (TLE). The anticonvulsant and adverse effects of TGB were examined in amygdala- or hippocampal-kindled rats and compared with those of other GABA uptake inhibitors (SKF89976A and NNC-711) and conventional antiepileptic drugs [AEDs: valproate (VPA) and carbamazepine (CBZ)]. In addition, the antiepileptogenic effects of TGB on amygdala kindling development were examined. TGB (2.5-40 mg/kg intraperitoneally, i.p.) had potent and dose-dependent anticonvulsant effects on both amygdala- and hippocampal-kindled seizures. The order of anticonvulsant potency of the three GABA uptake inhibitors tested was: NNC-711 > TGB > SKF-89976A and paralleled the in vitro GABA uptake efficacy. In addition, daily treatment with TGB 10 mg/kg for 10 days significantly retarded kindling development. Although adverse effects of TGB on motor systems were significantly less than those of VPA and CBZ, high toxic doses of TGB often caused EEG paroxysm and myoclonus. Our results indicate the clinical usefulness of TGB for treatment of drug-resistant TLE.

Suppressed Kindling Epileptogenesis and Perturbed BDNF and TrkB Gene Regulation in NT-3 Mutant Mice

In the kindling model of epilepsy, repeated electrical stimulations lead to progressive and permanent intensification of seizure activity. We find that the development of amygdala kindling is markedly retarded in mice heterozygous for a deletion of the neurotrophin-3 (NT-3) gene (NT-3+/− mice). These mice did not reach the fully kindled state (3rd grade 5 seizure) until after 28 ± 4 days of stimulation compared to 17 ± 2 days in the wild-type animals. The deficit in the NT-3+/− mice reflected dampening of the progression from focal to generalized seizures. The number of stimulations required to evoke focal (grade 1 and 2) seizures did not differ between the groups, but the NT-3 mutants spent a considerably longer period of time (13±3 days) than wild-type mice (2±1 days) in grade 2 seizures. As assessed by test stimulation 4–12 weeks after the 10th grade 5 seizure, kindling was maintained in the NT-3 mutants.In situhybridization showed 30% reduction of basal NT-3 mRNA levels and lack of upregulation of TrkC mRNA expression at 2 h after a generalized seizure in dentate granule cells of the NT-3+/− mice, whereas the seizure-evoked increase in brain-derived neurotrophic factor (BDNF) and TrkB mRNA levels was enhanced. These results indicate that endogenous NT-3 levels can influence the rate of epileptogenesis, and suggest a link between NT-3 and BDNF gene regulation in dentate granule cells.

The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures

The influence of 2-chloroadenosine, a non-metabolizable adenosine A 1 receptor agonist, was tested on the development of electrically kindled amygdala and on the seizure responses of fully kindled rats. Focal intra-amygdaloid injection of 2-chloroadenosine (1–10 nmol/0.5 μl) 20 min before applying the daily kindling stimulus prevented the development of the kindling process. The behavioural seizure score and the afterdischarge duration were reduced below their initial values. The antiepileptogenic effects of 1 and 10 nmol of 2-chloroadenosine were reversible 8–10 days after withdrawal of the drug. When 2-chloroadenosine was tested on fully developed stage 5 amygdala-kindled seizures, it increased the generalised seizure threshold in a dose-dependent manner. A maximum efficiency of 125% ( P<0.001) was achieved with 5 nmol and the median effective dose was 0.55 nmol. Higher doses resulted in the reduced anticonvulsant effect ( P<0.05). With the same daily stimulation, 2-chloroadenosine 5 nmol in 0.5 μl vehicle, significantly reduced the maximum seizure score by 90%, the afterdischarge duration by 88% and completely blocked the generalised seizure duration. The antiseizure activity of the drug lasted for 3 days. In conclusion, 2-chloroadenosine not only acts as an anticonvulsant against electrically induced kindled seizures as described here, and against audiogenic seizures, electroshock and a variety of chemical convulsants as described by others, it prevents the development of the epileptic state by kindling-stimulation, i.e., it is antiepileptogenic. We theorise here that this is due to its blockade of presynaptic glutamate release.

Role of serotonin receptor subtypes in the development of amygdaloid kindling in rats

The present study was conducted to identify serotonin (5-HT) receptor subtypes involved in the development of amygdala (AM) kindling. We used 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT), a 5-HT 1A agonist, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT 2 agonist, both of which were injected subcutaneously 15 min prior to each daily electrical stimulation to the rat AM. Treatment with 8-OH-DPAT (1 mg/kg) slightly suppressed behavioral and electrographic seizure development during the course of kindling. In contrast, DOI (1 mg/kg) strongly facilitated kindling development and reduced the number of stimulations needed to produce generalized seizures. These facilitatory effects of DOI were completely blocked by pretreatment with a 5-HT 2 antagonist ketanserin. The present results suggest that the activation of 5-HT 1A receptors can retard the development of AM kindling, whereas 5-HT 2 receptors play a facilitatory role in this developmental seizure process.

Amygdala-kindled seizures increase the expression of corticotropin-releasing factor (CRF) and CRF-binding protein in GABAergic interneurons of the dentate hilus

Kindling, a model of temporal lobe epilepsy, induces a number of neuropeptides including corticotropin-releasing factor (CRF). CRF itself can produce limbic seizures which resemble kindling in some aspects. However, tolerance to the convulsant effects of CRF develops rapidly. Hypothetically, this could be explained should seizures also induce the CRF-binding protein (CRF-BP), which has been postulated to restrict the actions of CRF. Therefore, in the present study, we used in situ hybridization to examine the effects of amygdala-kindled seizures on the mRNA levels of CRF and CRF-BP. Kindled seizures markedly elevated CRF and CRF-BP in the dentate gyrus of rats. CRF and CRF-BP were induced almost exclusively in GABAergic interneurons of the dentate hilus. The CRF and CRF-BP interneurons also expressed neuropeptide Y but not cholecystokinin. CRF appeared to have an excitatory role in the dentate gyrus as it decreased the afterhyperpolarization of dentate granule neurons. These results suggest that CRF may contribute to the development of amygdala kindling. However, the compensatory induction of CRF-BP may serve to limit the excitatory effects of CRF in the dentate gyrus.

Centrally acting histamine H1 antagonists promote the development of amygdala kindling in rats

Centrally acting histamine H1 antagonists promote the development of amygdala kindling in rats