Two types of neuroplasticities in the kindling phenomenon.

We stimulated the dorsal hippocampus of the rat with 2 Hz electrical stimulation to induce kindling seizures. As we reported previously using cats, pulse-number threshold (PNT), the number of stimulating-pulses required for the triggering of epileptic afterdischarge, decreased profoundly in the initial stage of the kindling process and the behavioral seizure stage (BSS) developed in the later stage. After the completion of kindling, a 4 week interval elevated PNT significantly compared to the value at the completion of kindling, whereas BSS showed no regression. These results suggest that there are two types of neuroplasticities which are independent of each other in the kindling phenomenon, one is the early-short type which is involved in the susceptibility of epileptic focus in hippocampus and the other is the late-long type which is involved in the full propagation of kindled seizures to the extra-limbic area.     

Behavioral effects of high frequency electrical stimulation of the hippocampus on electrical kindling in rats

Experiments were designed to evaluate the effects of high frequency electrical stimulation (HFS) applied in ventral hippocampus during the hippocampal kindling process, as well as on the expression of fully kindled seizures and the refractoriness for subsequent convulsions during their postictal period. Male Wistar rats, stereotactically implanted in both ventral hippocampus, received daily bilateral HFS (pulses of 60 micros width at 130 Hz at subthreshold current intensity) during 1h immediately after each kindling stimulation (1s train of 60 Hz biphasic square waves, each 1 ms) during 40 days or until the kindled state was achieved. Rats were classified as follows: (a) Responder animals, who required low current intensity for HFS (208+/-38.2 microA), did not show progress of the kindling process and remained in stages II and III seizures. (b) Nonresponders rats, in which the current intensity for HFS was higher (434.5+/-51.7 microA), developed the kindling process as the kindling control group. When HFS was applied before the kindling stimulation in fully kindled rats, animals presented a reduced expression of the fully kindled seizures (nonresponders animals) and an enhanced refractoriness for subsequent seizures during the postictal period (kindling control and nonresponder animals). There was no correlation between the area where the HFS was applied and the effects induced. It was concluded that HFS at 130 Hz in ventral hippocampus is able to modify the epileptogenesis induced by the hippocampal kindling process and the refractoriness to subsequent seizures during the postictal period in rats.     

氟桂利嗪对杏仁核点燃鼠海马Bax mRNA表达的影响

目的:观察氟桂利嗪对杏仁核点燃鼠癎性发作及海马促凋亡基因Bax mRNA表达的影响。方法:建立杏仁核点燃模型,予不同剂量氟桂利嗪灌喂点燃鼠。原位杂交法检测鼠脑海马Bax mRNA表达,图像分析软件测量阳性细胞平均吸光度。结果:正常大鼠海马存在少量Bax mRNA表达阳性细胞,点燃鼠海马各区Bax mRNA表达阳性细胞数及平均吸光度增加,氟桂利嗪处理后平均吸光度下降与剂量有关。结论:氟桂利嗪具有抗癫癎效应和拮抗点燃鼠海马Bax mRNA表达的作用。     

Morphometric effects of intermittent kindled seizures and limbic status epilepticus in the dentate gyrus of the rat

The effect of recurrent seizures on the hippocampus has been controversial for many years. To determine the effect different seizure paradigms had on the structure of the dentate gyrus, we conducted histological studies on the dentate gyrus (DG) from three groups of rats: (1) those that had experienced 1500 intermittent kindled seizures; (2) those that had experienced a single episode of limbic status epilepticus (SE); and (3) control rats that had been implanted with electrodes. When compared to controls the DG of SE rats was overall slightly, but non-significantly, smaller, but the DG of rats with 1500 kindled seizures was significantly larger. The decrease of size following SE was attributable to a significant atrophy of the molecular layer. The increase in area associated with kindling was the result of an enlargement of the molecular layer and the hilus. Absolute neuronal counts showed a decrease in the hilus after SE but no change following kindling, but both groups had decreased neuronal densities in the hilus when compared to controls. The decreased density after SE was secondary to neuronal loss, but the decrease in neuronal density following kindling was the result of the expansion of the hilar neuropil without change in the number of neurons. This study extends our previous findings in Ammon's horn and indicates that SE induces significant neuronal loss, but numerous intermittent kindled seizures have no effect on neuronal numbers in the DG.     

Regional brain concentrations of neurotensin following amygdaloid kindled and cortical suprathreshold stimulation-induced seizures in the rat

There is now considerable evidence that putative neuropeptides are involved in epileptogenic processes. Neurotensin (NT), which affects neuronal excitability in many parts of the CNS, is potentially relevant to the kindling phenomenon, but has not previously been studied in the kindling model of epilepsy. This study compared the short term (24 hr) and long term (3 wk) effects of amygdaloid kindled seizures, and the short term effects of non-kindled seizures (cortical suprathreshold stimulation-induced seizures, STS), on the concentrations of NT in 13 brain regions in the rat. Whereas kindled seizures produced neither short term nor long term changes in NT, the STS resulted in altered NT concentrations, with a decrease in the cortex and increases in the hippocampus and cervical spinal cord. These results reveal seizure-type specific changes in NT in 3 brain regions; and indicate that although NT does not appear to be involved in kindling, it may be important in the neurochemical mechanisms underlying STS.     

Kindling in developing animals: interactions between ipsilateral foci.

In adult rats, concurrent kindling of two limbic sites, alternating stimulation to each site, often results in the retarding of kindling at one or both sites. This inhibitory interaction between two limbic kindling foci is termed kindling antagonism and occurs irrespective of whether the sites are contralateral or ipsilateral. We have previously shown that kindling antagonism does not occur when 16- to 17-day-old rat pups are concurrently kindled in the hippocampus and contralateral amygdala or between the two amygdalae. In this study, adult and 16- to 17-day-old rats were concurrently kindled in the hippocampus and ipsilateral amygdala to determine if the local intrahemispheric mechanisms suppressing multiple kindled foci are age-dependent. Kindling antagonism occurred in 7 out of 10 adult rats. In contrast, in rat pups, kindling development was not suppressed. Concurrent kindling of the two limbic sites enhanced the development of severe seizures. Two 16- to 17-day-old rats receiving alternating stimulations exhibited spontaneous seizures. The age-specific failure of both inter- and intrahemispheric mechanisms to suppress the development of multiple kindling foci may explain the high incidence of multifocal seizures in the immature CNS.     

Hippocampal Nogo-A and neo-Timm's staining in amygdala kindling rats

PURPOSE: Nogo-A, one of the axon regeneration inhibitors, has been shown to be up-regulated in both the experimental and human temporal lobe epilepsy. However, the role of Nogo-A in mossy fiber sprouting (MFS) relative to epileptogenesis is unknown. This work was designed to examine the relationship of the hippocampal Nogo-A protein expression with MFS during the development of amygdala kindling. METHODS: Using immunohistochemistry and neo-Timm's histological procedures, we evaluated the distribution and density of Nogo-A and Nogo-66 receptor (Ng-R) expression and MFS in the bilateral hippocampus of amygdala kindling rats. RESULTS: Nogo-A expression in the ipsilateral hippocampus gradually increased with the development of kindling in the sector CA2-3. In contrast, no increased Nogo-A expression was observed in the contralateral hippocampus as the rats advanced to stage 5 kindled seizures. Furthermore, poorer Nogo-A and Nogo-66 receptor (Ng-R) expression were observed in the dentate granule cells as aberrant MFS occurred. CONCLUSIONS: In amygdala kindling rats, generalized stage 5 seizures were not associated with increased Nogo-A expression in the contralateral hippocampus supporting the concept that seizures by themselves do not induce Nogo-A expression. Furthermore, in the ipsilateral hippocampus, the expression of Nogo-A relative to MSF suggests that this protein may partially control aberrant synaptic reorganization during epileptogenesis.     

Secondary generalization of hippocampal kindled seizures in rats: examining the role of the piriform cortex

A primary feature of epilepsy is the potential for focal seizures to recruit distant structures and generalize into convulsions. Key to understanding generalization is to identify critical structures facilitating the transition from focal to generalized seizures. In kindling, development of a primary site leads progressively to secondarily generalized convulsions. In addition, subsequent kindling of a secondary site results in rapid kindling from that site, presumably because of its facilitated access to the primary kindled network. Here, we investigated the role of the piriform cortex in convulsive generalization from a secondary site kindled in the hippocampus after primary site amygdala kindling. In a necessarily complicated design, rats initially experienced forebrain commissurotomy to lateralize the experiment to one hemisphere. Then the amygdala was kindled and, 3 weeks later, it was electrically-triggered into status epilepticus, which destroyed the ipsilateral piriform cortex. This experience occurred several days before secondary site kindling of the dorsal hippocampus. In rats with complete piriform cortex loss, there was no disruption in kindling or convulsive seizure expression from the hippocampus. However, when damage also involved parts of the perirhinal, insular and entorhinal cortices, convulsive expression was blocked. Although other evidence suggests that piriform lesions affect generalization of primary site kindling, the present study shows that they do not alter secondary site kindling in the dorsal hippocampus. The additional involvement of parahippocampal cortical areas in convulsive expression suggests an important functional association between these cortical regions and the hippocampus in seizure propagation and clinical expression.     

Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats

Summary: Purpose: Based on data from brain biopsy samples of patients with pharmacoresistant partial epilepsy, overexpression of the multidrug transporter P‐glycoprotein (PGP) in brain capillary endothelium has recently been proposed as a potential mechanism of resistance to antiepileptic drugs (AEDs). We examined whether PGP is overexpressed in brain regions of amygdala‐kindled rats, a widely used model of temporal lobe epilepsy (TLE), which is often resistant to AEDs. Methods: Rats were kindled by stimulation of the basolateral amygdala (BLA); electrode‐implanted but nonkindled rats and naive (not implanted) rats served as controls. PGP was determined by immunohistochemistry either 1 or 2 weeks after the last kindled seizure, by using a monoclonal anti‐PGP antibody. Six brain regions were examined ipsi‐ and contralateral to the BLA electrode: the BLA, the hippocampal formation, the piriform cortex, the substantia nigra, the frontal and parietal cortex, and the cerebellum. Results: In both kindled rats and controls, PGP staining was observed mainly in microvessel endothelial cells and, to a much lesser extent, in parenchymal cells. The distribution of PGP expression across brain regions was not homogeneous, but significant differences were found in both the endothelial and parenchymal expression of this protein. In kindled rats, ipsilateral PGP expression tended to be higher than contralateral expression in several brain regions, which was statistically significant in the piriform cortex and parietal cortex. However, compared with controls, no significant overexpression of PGP in capillary endothelial cells or brain parenchyma of kindled rats was seen in any ipsilateral brain region, including the BLA. For comparison with kindled rats, kainate‐treated rats were used as positive controls. As reported previously, kainate‐induced seizures significantly increased PGP expression in the hippocampus and other limbic brain regions. Conclusions: Amygdala‐kindling does not induce any lasting overexpression of PGP in several brain regions previously involved in the kindling process. In view of the many pathophysiologic and pharmacologic similarities between the kindling model and TLE, these data may indicate that PGP overexpression in pharmacoresistant patients with TLE is a result of uncontrolled seizures but not of the processes underlying epilepsy. It remains to be determined whether transient PGP overexpression is present in kindled rats shortly after a seizure, and whether pharmacoresistant subgroups of kindled rats exhibit an increased expression of PGP. Furthermore, other multidrug transporters, such as multidrug resistance–associated protein, might be involved in the resistance of kindled rats to AEDs.     

Increases in mRNA levels for Talpha1-tubulin in the rat kindling model of epilepsy

The expression of mRNA for Talpha1-tubulin, a cytoskeletal protein, was studied in the rat kindling model of epilepsy. The Talpha1-tubulin mRNA level increased significantly in the dentate gyrus (DG) and CA3 of hippocampus ipsilateral to stimulation from 8 h to 4 weeks after amygdaloid kindled seizures. The peak increase was observed at 1 week after the last seizures both in the DG and CA3. These results suggest that the microtubule formation contributes to synaptic remodeling and reorganization of neural networks, which may be based on the kindling-inducing epileptogenesity.     

The insular but not the perirhinal cortex is involved in the expression of fully-kindled amygdaloid seizures in rats.

We have previously reported an important excitatory role of the perirhinal cortex (PRC) in rat kindling development using an immunohistochemistry technique. In this study, we investigated the roles of the PRC and the insular cortex (INS) located rostral to the PRC, in fully-kindled amygdaloid seizures, using a microinjection technique in the rat kindling model of epilepsy. Following the establishment of daily kindling, we investigated the effects of microinjections of procaine hydrochloride, 2-amino-5-phosphonovalerate (APV; an N-methyl-D-aspartate (NMDA) receptor antagonist) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX; an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor antagonist). Microinjections of these drugs into the ipsilateral PRC did not suppress kindled seizures. The possibility is that the process of kindling development forms novel seizure-generalization pathways that do not require further activation of the PRC. On the other hand, procaine and APV injected into the ipsilateral INS significantly suppressed kindled seizures. The manner of suppression appeared to be 'all or none'. It is therefore possible that at least the activation of NMDA receptors in the INS is necessary to express generalized kindled amygdaloid seizures.     

An ontogenetic study of kindling using rapidly recurring hippocampal seizures

The present study investigated the acquisition and retention of kindling in immature rats. Postnatal (PN) 7-28-day-old rats were electrically kindled in the ventral hippocampus. Ten-second, 20-Hz stimulus trains were delivered every 5 min for 6 h on one day (short interval rapidly recurring hippocampal seizures, RRHS) or every 30 min for 9 h on each of two consecutive days (long interval RRHS). Afterdischarge durations (ADD) and behavioral seizure scores (BSS) were recorded following each stimulation. Animals of all ages kindled with both short and long interval RRHS, as manifested by lengthening of ADD and increasing BSS. With short interval RRHS, the course of kindling was erratic; with long interval RRHS, kindling proceeded smoothly over both test days. In PN 14-28 rats, the degree of kindling obtained on the first day of long interval RRHS was retained at the start of the second experimental day. In contrast, PN 7 rats showed a transient decrease in ADD and BSS from day 1 to day 2. Afterdischarge thresholds declined with maturation. Among the PN 14-28 animals, younger rats exhibited longer seizures at the outset of kindling and proceeded through kindling faster. Once established, kindled motor seizures also occurred with 2-s, 50-Hz stimulus trains. We conclude that rapid kindling occurs at all ages; however, PN 7 rats are less capable of retaining the kindling effect than are older rats.     

Increases in mRNA levels for Tα1-tubulin in the rat kindling model of epilepsy

The expression of mRNA for Tα1-tubulin, a cytoskeletal protein, was studied in the rat kindling model of epilepsy. The Tα1-tubulin mRNA level increased significantly in the dentate gyrus (DG) and CA3 of hippocampus ipsilateral to stimulation from 8 h to 4 weeks after amygdaloid kindled seizures. The peak increase was observed at 1 week after the last seizures both in the DG and CA3. These results suggest that the microtubule formation contributes to synaptic remodeling and reorganization of neural networks, which may be based on the kindling-inducing epileptogenesity.     

Changes in cholecystokinin mRNA expression after amygdala kindled seizures: an in situ hybridization study

Cholecystokinin (CCK) can be a potent anticonvulsant neuropeptide in certain seizure models. Therefore, we examined whether seizures produced by electrical kindling of the amygdala or electroconvulsive seizures (ECS) would affect the expression of CCK mRNA in rat brain. Following a single kindled seizure, CCK mRNA expression was decreased about 20–58% in the amygdala. In contrast, after multiple consecutive kindled seizures, CCK mRNA expression was increased in the amygdala, cerebral cortex, CA1 pyramidal cell layer of the hippocampus and dentate hilus. A single ECS produced no effect on CCK mRNA expression, but multiple ECS increased expression in the interneurons of the hippocampus 24 h after the last seizure. Since seizures produced by ECS can be anticonvulsant to further ECS or kindled seizures, the CCK increases in the hippocampus may represent a compensatory anticonvulsant adaptation observed in both models. Overall, the kindling-induced alterations in CCK expression appear to be more complex involving multiple brain regions and distinct temporal properties.     

Kindling causes changes in the composition of the astrocytic cytoskeleton.

Changes in the astrocytic cytoskeleton were examined in amygdala kindled rats using immunocytochemical techniques. One week following kindling, there was a dramatic increase in immunoreactivity to glial fibrillary acidic protein and vimentin in astrocytes throughout amygdala, pyriform cortex and hippocampus bilaterally. Since these changes occurred in anatomical sites involved in the propagation of kindled seizures, the observed cytoskeletal reorganization in astrocytes may signify important functional alterations in the kindled brain.     

Anticonvulsive and antiepileptogenic effects of levetiracetam in the audiogenic kindling model

Purpose: To study anticonvulsive and antiepileptogenic effects of singe levetiracetam (LEV) administration in the model of audiogenic kindling.
Methods: Rats of Krushinsky-Molodkina (KM) strain genetically susceptible to severe audiogenic seizures received one intraperitoneal injection of saline, low (6 mg/kg) or high (50 mg/kg) dose of LEV before or after audiogenic kindling. One hour postinjection, an audiogenic seizure was induced to assess anticonvulsive effect of LEV in nonkindled and kindled rats. To examine antiepileptogenic activity of LEV, nonkindled rats injected with the drug or saline were kindled with repeated sound stimulations. Audiogenic kindling development manifested in an appearance and progressive prolongation of an additional seizure phase, post-tonic–clonus. The latency and duration of audiogenic seizures and the duration of every seizure phase (running, tonic, post-tonic–clonic) were measured.
Results: One hour posttreatment, LEV dose-dependently lengthened the latency and reduced the duration of audiogenic seizures in both nonkindled and kindled rats. The seizure shortening resulted from selective suppression of tonic and kindled post-tonic–clonic phases. The dose of 50 mg/kg completely blocked tonic and clonic convulsions 1 h postinjection. The anticonvulsive effect of LEV was more pronounced in kindled than in nonkindled rats. Single LEV injection in the dose of 50 mg/kg prior audiogenic kindling significantly suppressed subsequent kindling progression indicating profound antiepileptogenic potency of the drug.
Conclusions: The present study shows that LEV exerts both short-lasting anticonvulsive effect on audiogenic seizures and very long-lasting antiepileptogenic effect on audiogenic kindling. Remarkably, a single injection of LEV is enough to significantly suppress kindling progression in KM rats.     

Amygdala-kindled and electroconvulsive seizures alter hippocampal expression of the m1 and m3 muscarinic cholinergic receptor genes

Expression of m1 and m3 muscarinic cholinergic receptors mRNAs was examined in rat hippocampus following either: (1) kindling to five Stage 5 amygdala-kindled seizures; or (2) eight electroconvulsive shock (ECS) seizures. Twenty-four hours after the last seizure of either type, there was a significant decrease in both m1 and m3 mRNAs in CA1, CA3 and the dentate gyrus subfields of the hippocampus. Twenty-eight days after the last seizure of either type, there was a significant increase in m1 mRNAs in CA1, CA3, and the dentate gyrus; for m3 mRNAs, there was a significant increase in CA3 28 days after the last ECS seizure, and in CA1 and CA3 28 days after the last kindled seizure. These results suggest that seizures alter the cholinergic system in the hippocampus, and that some of the alterations are very long-lasting.     

Expression of an embryonic intermediate filament protein in amygdaloid kindled rats

Amygdaloid kindling is well known as an experimental model of temporal lobe epilepsy. However, the mechanism of kindling epileptogenesis remains unclear. To examine the remodelling process in kindling, we performed immunohistochemistry of nestin, an embryonic intermediate neurofilament protein, in amygdaloid kindled rats. In rats expressing focal seizures (kindling stage C3), nestin immunoreactive cells (NIC) were detected at ipsilateral piriform cortex (PC) and ipsilateral perirhinal cortex (PRh), and at PC bilaterally in fully kindled rats expressing secondary generalized seizures (kindling stage C5). Double staining with glial fibrillary acidic protein revealed that almost all reactive astrocytes at PC express nestin immunoreactivity. These results suggest that glial NIC may participate in the remodelling process at the PC and PRh areas. This is the first report of nestin expression in kindling and suggests that glial nestin at PC and PRh may play a significant role in permanent epileptogenesis in kindling.     

Effects of some antiepileptics on septal-kindled seizures in rats

PURPOSE: The present study was performed to understand the characteristics of septal kindling in rats, especially the efficacies of antiepileptic drugs in comparison with amygdala kindling. METHODS: Under pentobarbital anesthesia, rats were fixed to a stereotaxic apparatus, and electrodes were implanted into the right frontal cortex, the hippocampus, the lateral septal and the amygdala. Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. Bipolar stimulation was applied to the lateral septal or the amygdala every day until a generalized seizure was obtained. Carbamazepine (CBZ), zonisamide (ZNS) and clobazam (CLB) were orally administered to fully kindled rats. RESULT: A considerable number of stimulations was required to establish septal-kindled seizures. In addition, wet dog shakes were observed during the septal kindling procedure, different from amygdala kindling. The oral administration of CBZ, ZNS and CLB caused a dose-dependent inhibition both of seizure stage and after-discharge (AD) duration of septal-kindled seizures. CBZ and ZNS caused a more potent inhibition of septal-kindled seizures than amygdala kindled seizures, whereas CLB inhibited both septal and amygdala kindled seizures to almost the same extent. CONCLUSION: Septal kindling was confirmed to possess some characteristics, which were evidently different from that of amygdala kindling. In addition, it was demonstrated that septal kindling was also available as a model for the evaluation of antiepileptic drugs.